Attenuated bacteria useful in vaccines

ABSTRACT

The invention provides strains of bacteria, especially enterotoxigenic  E. coli , attenuated by mutations in the genes encoding enterotoxins (LT, ST, EAST1) and optionally further attenuated by deletion of additional chromosomal genes. In addition the invention provides strains of attenuated bacteria expressing immunogenic but non-toxic variants of one or more of these enterotoxins. These bacteria are useful as a vaccine against diarrhoeal disease.

This application is a continuation of application Ser. No. 10/489,273,filed Sep. 22, 2004 and now U.S. Pat. No. 7,943,122; which is the U.S.national phase of Int'l Application No. PCT/GB02/04164, filed on 11 Sep.2002. The entire contents of these applications are incorporated hereinby reference.

The invention relates to attenuated bacteria useful in vaccines.

BACKGROUND TO THE INVENTION

The principle behind vaccination is to induce an immune response in therecipient, thus providing protection against subsequent challenge with apathogen. This may be achieved by inoculation with a live attenuatedstrain of the pathogen, i.e. a strain having reduced virulence such thatit does not cause the disease caused by the virulent pathogen whilestill stimulating a broad immune response.

Using modern genetic techniques, it is now possible to constructsite-directed attenuated bacterial strains in which stable attenuatingdeletions have been created. A number of site-directed mutants ofSalmonella have been created using this type of technology (2, 7, 9, 14,19, 35, 36, 37). Mutations in a large number of genes have been reportedto be attenuating, including the aro genes (e.g. aroA, aroC, aroD andaroE (15, 18)), pur, htrA (4), ompR, ompF, ompC (2), galE (14), cya, crp(7), phoP (13, 19), rfaY (48), dksA (48), hupA (48), sipC (48) and clpB(48).

One class of bacterium that has been attenuated by such modern genetictechniques is enterotoxigenic Escherichia coli (ETEC), which causesdiarrhoea. The virulence of (ETEC) strains depends on their expressionof fimbrial colonization factor antigens (CFAs) which allow them toattach to and colonize the mucosal surface of the small intestine oftheir host species. Human adapted ETEC strains express a number of CFAs,the most frequently occurring of which are CFA/I, CFA/II (comprising CS3expressed with either CS1 or CS2) and CFA/IV (comprising CS6 expressedalone or with either CS4 or CS5). Depending on the geographic location,CFA/I, CFA/II and CFA/IV account for between 50% and 80% of ETECstrains. Many other CFAs have been described, but each of them is foundin only a small proportion of ETEC strains (33). Evidence indicates thatanti-CFA immune responses are important for protection against ETECdisease (6, 24, 28, 29, 30).

Colonization of the small intestine is accompanied by the secretion ofenterotoxins. Two types of enterotoxins have been identified in ETECstrains, the heat labile toxin (LT) and the heat stable toxin (ST). LTis highly homologous in structure to the cholera toxin, a multi-subunitprotein of the form AB₅. The A subunit is the active component of thetoxin, which functions to increase the activity of adenylate cyclase.This is delivered into host cells by the B subunits, which bind togangliosides on the cell surface. ST is a small (19 amino acid)non-immunogenic polypeptide that has guanylate cyclase stimulatingactivity. In addition, it has been demonstrated recently that a largeproportion of ETEC strains also produce EAST1, a heat-stable toxinsimilar in size and mode of action to ST but different in sequence,which was originally identified in enteroaggregative E. coli strains(34).

It has been proposed that derivatives of ETEC strains, which have lostthe ability to produce toxins, may be effective live vaccines againstvirulent isolates. A derivative of a wild-type ETEC strain, E1392/75,that has spontaneously lost the ST and LT activities but which continuesto express CFA/II was identified and designated E1392/75-2A (5). Inhuman volunteer studies, oral vaccination with 2×10¹⁰ cfu E1392/75-2Agave 75% protection against challenge with a toxin-expressing ETEC froma different serotype but which expressed the same CFAs (reviewed by(30)). However, approximately 15% of vaccinees experienced milddiarrhoea as a side effect of the vaccine. It was concluded that furtherattenuation of this strain was required before it could be consideredfor use as a live vaccine against ETEC infections.

Two derivatives of E1392/75-2A were generated by targeted deletion ofpotential attenuating genes and evaluated in clinical trials (32, 38).It was demonstrated that both of the derivatives (PTL002, ΔaroC/ΔompR,and PTL003, ΔaroC/ΔompC/ΔompF) were attenuated when compared to theparent strain and caused no clinical symptoms in volunteers who ingestedup to 5×10⁹ cfu of freshly harvested live organisms. All volunteersreceiving the maximum dose of these candidate vaccines generatedspecific immune responses against the CFA/II antigen expressed by thestrains.

SUMMARY OF THE INVENTION

An effective vaccine against ETEC must immunize against CFA/I, CFA/IIand CFA/IV as a minimum, and therefore attenuated strains expressing allof these antigens are required. Thus, it is required that the genesexpressing the toxins LT, ST and EAST1 are inactivated or deleted fromstrains expressing all of these CFAs. Toxin minus strains havepreviously been suggested as a starting point for developing a liveattenuated multi-strain vaccine against ETEC (Chatfield, 38). There was,however, no explanation in Chatfield as to how such strains might begenerated.

We have now found that there are particular difficulties associated withthe generation of a strain expressing CFA/I or a strain expressing CS5and CS6 from which the toxin genes, especially the ST gene, have beendeleted. We have devised a novel strategy and suicide vector forovercoming these difficulties and producing toxin minus forms of thesestrains.

Without wishing to be bound by this theory, we believe that the reasonthat ST minus forms of strains expressing CFA/I or CS5 and CS6 weredifficult to generate was that the CFA/CS genes are closely linked tothe ST gene and are on the same plasmid. In a global review ofepidemiological studies in which sufficient data had been collected (33)it is reported that of 204 CFA/I expressing strains, all 204 of themexpressed ST, either alone (149/204) or in combination with LT (55/204).Further, more recent studies have confirmed this finding, e.g. Qadri etal (22), where all of 87 CFA/I expressing strains isolated in a two yearperiod in Bangladesh expressed ST, either alone or in combination withLT. No strains were identified which expressed CFA/I and LT alone,suggesting an extremely tight genetic linkage between the ST and CFA/Iloci. Numerous scientific papers document the close linkage betweenCFA/I and ST genes in ETEC strains, to the extent that whenever aneffort has been made to derive a strain which has lost one of theseloci, the other has always been lost concurrently. In no instance ofwhich we are aware has it been possible to separate the two loci bydeletion or inactivation of the ST gene and produce a strain that stillexpresses CFA/I (42-46).

The invention provides a bacterial cell which expresses colonizationfactor antigen CFA/I from a native plasmid but does not express heatstable toxin (ST). The invention also provides a bacterial cell whichexpresses colonization factor antigen CS5 from a native plasmid and/orexpresses colonization factor antigen CS6 from a native plasmid, butdoes not express heat stable toxin (ST). The LT gene and the EAST1 genemay also be deleted or inactivated in the cells of the invention. Thecells generally contain further attenuating mutations, such as mutationsin each of the aroC, ompF and ompC genes, in order to make themacceptable for use in vaccines.

The cells of the invention may be genetically engineered to express aheterologous antigen, such as a non-toxic component or form of LT, or acolonization factor antigen (CFA). Such cells induce an immune responseagainst the heterologous antigen as well as the native antigens andhence improve the protection provided by a vaccine.

The invention includes a vaccine against diarrhoea containing the cellsof the invention. Preferably, the vaccine includes a blend of differentcells which between them carry all the most common CFAs, namely CFA/I,CFA/II and CFA/IV.

Furthermore, the invention provides a suicide vector and a method whichmakes possible the reliable and rapid isolation of the cells of theinvention and other bacterial cells containing deleted, inactivated orreplaced genes. The vector represents an improvement over known suicidevectors in that it allows more specific and more reliable targeting thanknown vectors. The vector is less than 5 kb in size (e.g. from 2.5 to 5kb or from 2.5 to 4 kb) and comprises the sacB region which codes for aproduct that is toxic to bacteria when grown on sucrose, in which regionthe IS1 insertion sequence is deleted or inactivated. The small size ofthe vector and the absence of the IS1 insertion sequence help to preventthe vector from targeting to the wrong place in the cellular DNA.

DETAILED DESCRIPTION OF THE INVENTION

Bacteria Useful in the Invention

The bacterial cells of the invention are generally derived fromenterotoxigenic E. coli (ETEC) cells by deletion or inactivation of theST gene and optionally other toxin genes. As mentioned above, ETEC is aclass of E. coli that cause diarrhoea. They colonise the smallintestine. They can be isolated from human clinical samples, typicallystools produced whilst suffering from diarrhoea. A standard ETEC strainis H10407, deposited at the ATCC under catalogue #35401.

Infections of ETEC are the single most frequent cause of travellersdiarrhoea, causing 3-9 million cases per year amongst visitors todeveloping countries. In endemic areas, ETEC infections are an importantcause of dehydrating diarrhoea in infants and young children, resultingin up to 400,000 deaths a year, predominantly in this age group. Indeveloping countries, the incidence of ETEC infections leading toclinical disease decreases with age, indicating that immunity to ETECinfection can be acquired. In contrast, naive adults from industrializedcountries who visit endemic areas are highly susceptible to ETECinfections. However, with prolonged or repeated visits to endemic areassusceptibility to ETEC infections diminishes, suggesting that a liveattenuated approach to ETEC vaccination may prove successful.

A vaccine to protect against ETEC diarrhoea in humans must provideprotection against the seven major colonization factors and, as aminimum, the heat labile toxin (LT) to ensure that protection againstdifferent strains is obtained. In order to achieve this, the sameattenuations could be made in a range of different ETEC strains, eachwith a different colonization factor. This would involve deleting thetoxins from all such strains. The present invention provides a panel ofsuitable strains from which all toxin genes have been completely deletedwhich can provide the starting point for the generation of amulti-strain vaccine. Alternatively, it may be possible to expressmultiple colonization factors in a smaller number of strains from whichthe toxins have been similarly deleted.

Toxin-deleted strains of the present invention were derived fromwild-type clinical isolates obtained from a long-term epidemiologicalstudy carried out in Egypt by scientists at the US Navy NAMRU3 facilityin Cairo. A list of the strains provided is given in the followingtable.

Strain Code Phenotype CFA LT ST EAST1 WS-1858B A O71:H- CFA/I − + +WS-4437A B O128:H12 CFA/I − + − WS-6117A C O153:H45 CFA/I − + + WS-2560BD O25:H- CS4, CS6 + + + WS-2773E E O39:H12 CS5, CS6 + + + WS-4150D FO6:H16 CS2, CS3 + − − WS-6170A G O17:H18 CS2, CS3 − + − WS-3504D HO141:H5 CS2, CS3 + + + WS-3517A I O6:H- CS2, CS3 − + + WS-2252A JO15:H18 CS4, CS6 + + + WS-2511A K O4:H- CS4, CS6 − + + WS-2556A L O6:H1CS4, CS6 − + + WS-4046A M O39:H- None identified + − N.D.

It will be clear to those skilled in the art that other strains may beequally suitable as a starting point for the generation of atoxin-deleted, attenuated multi-strain vaccine.

The strains with the codes A, B, E, H & J were attenuated by thespecific removal of all known toxin genes and then further manipulated,as described in the accompanying Examples. Resulting toxin-minus strainsand strain PTL003 described above were deposited by Acambis ResearchLimited of Peterhouse Technology Park, 100 Fulbourn Road, Cambridge, CB1 9PT, United Kingdom with the European Collection of Cell Cultures(ECACC), CAMR, Salisbury, Wiltshire SP4 OJG, United Kingdom on 3 Sep.2001 (accession numbers 010903**) or 29 Aug. 2002 (accession numbers020829**) in accordance with the Budapest Treaty. The characteristics ofthe strains and the accession numbers of those which were deposited areas follows:

Accession Antibiotic CS Strain Parent Strain number LPS:flagellinResistance proteins Regulator Toxin Genes Example E1392/75-2A E1392/75N/A O6:H16 Strep CS1 CS3 rns None PTL003 E1392/75-2A 01090302 O6:H16Strep CS1 CS3 rns None (submitted as ACM2005) ACAM2008 PTL003 02082965O6:H16 None CS1 CS3 rns None WS-2773E N/A N/A O39:H12 None CS5 CS6 ?csvR ST EAST LT 3 (strain E) WS-2773E 01090305 O39:H12 None CS5 CS6 ?csvR None WS-2773E- (submitted as Tox ACM2002) minus ACAM2006WS-2773E-Tox minus N/A O39:H12 None CS5 CS6 ? csvR None ACAM2012*ACAM2006 02082968 O39:H12 None CS5 CS6 ? csvR None WS-3504D N/A N/AO141:H5 Amp CS2 CS3 rns EAST 4 (strain H) WS-3504D 01090304 O141:H5 AmpCS2 CS3 rns None WS-3504D- (submitted as Tox ACM2003) minus ACAM2007WS-3504D-Tox minus 02082964 O141:H5 None CS2 CS3 rns None WS-1858B N/AN/A O71:H- Amp/Tmp/Smz CFA/I rns ST EAST 2 (Strain A) WS-1858B N/AO71:H- Amp/Tmp/Smz CFA/I rns None WS-1858B- Tox minus ACAM2010WS-1858B-Tox minus 02082967 O71:H- None CFA/I rns None WS-2252A N/A N/AO15:H18 None CS4 CS6 cfaD ST EAST LT 4 (Strain J) WS-2252A 01090306O15:H18 None CS4 CS6 cfaD None WS-2252A- (submitted as Tox ACM2004)minus ACAM2009 WS-2252A-Tox minus 02082966 O15:H18 None CS4 CS6 cfaDNone WS-2511A N/A N/A O4:H- None CS4 CS6 cfaD ST EAST × 2 N/A (Strain K)Strain K WS-2511A-Tox minus N/A O4:H- None CS6 cfaD ST EAST × 2*ACAM2006 contains a lysogenic phage in its chromosome - ACAM2012 is aderivative of ACAM2006 from which a large part of the genome, includingseveral genes critical for phage assembly, have been deleted.

In addition to the strains in the above table, a toxin minus derivativeof strain B described in Example 2 below was deposited under accessionnumber 01090303.

The invention includes “descendents” of these deposited cells. A“descendent” is any cell derived from a deposited cell. The descendentsof a deposited cell include cells with one or more further attenuatingmutations, such as the mutations described below. Descendents alsoinclude cells which have been engineered to express heterologousantigens, such as the heterologous antigens described below.

Although the bacteria of the invention are generally E. coli bacteria,other types of bacteria may be used. A plasmid in accordance with theinvention may be constructed by deleting or inactivating the ST gene ina plasmid native to enterotoxigenic E. coli, and then transferring theresulting plasmid to another bacterium. In this way, the other bacteriummay be made to carry the CFA/I antigen or the CS5 and CS6 antigens.

The bacteria that are used to make the vaccines of the invention aregenerally those that infect by the oral route. The bacteria may be thosethat invade and grow within eukaryotic cells and/or colonize mucosalsurfaces. The bacteria are generally gram negative but in someembodiments gram positive bacteria may be used. The bacteria aregenerally pathogens.

The bacteria used may be from the genus Escherichia, Salmonella,Shigella or Vibrio.

Other than E. coli, examples of the species, of bacteria that can beused in the invention are Salmonella typhimurium—the cause ofsalmonellosis in several animal species; Salmonella typhi—the cause ofhuman typhoid; Salmonella enteritidis—a cause of food poisoning inhumans; Salmonella choleraesuis—a cause of salmonellosis in pigs; andSalmonella dublin—a cause of both a systemic and diarrhoel disease incattle, especially of new-born calves.

Strains of E. coli and Salmonella are particularly useful in theinvention. Salmonella are potent immunogens and are able to stimulatesystemic and local cellular and antibody responses. In particular anattenuated strain of Salmonella typhi is preferred for use in theinvention. Preferred Salmonella typhi strains for use in the presentinvention include CVD908-htrA (ΔaroC ΔaroD ΔhtrA) and CVD908 (ΔaroCΔaroD) (49). Strains of E. coli other than ETEC that may be used includeenteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC) andenterohemorrhagic E. coli (EHEC).

As used herein, references to a “native” plasmid which expresses CFA/Ior CS5/6 mean a plasmid which exists in wild-type cells, for exampleETEC cells isolated from a person with diarrhoea. They exclude a plasmidconstructed in the laboratory for expression of CFA/I or CS5/6. In acells of the invention, the ST gene in the native plasmid is deleted orinactivated. However, the CFA/I or CS5/6 gene in the plasmid isfunctional, i.e. it expresses CFA/I or CS5/6.

Further Mutating the Bacteria

In order for the bacteria to be used in a vaccine, they must generallybe further attenuated. The attenuation may, for example, be broughtabout by deleting or inactivating one or more of the following genes:aroA, aroC, aroD, aroE, pur, htrA, ompC, ompF, ompR, cya, crp, phoP,surd, rfaY, dksA, hupA, sipC and clpB. Preferred combinations of genesinclude:

-   -   at least one aro gene (e.g. aroA, aroC, aroD or aroE) and at        least one omp gene (e.g. ompC, ompF or ompR);    -   at least one aro gene (e.g. aroA, aroC, aroD or aroE) and the        htrA gene;    -   aroC, ompF and ompC.

The further attenuating mutations may be introduced using the suicidevector and methods of the invention or by methods known to those skilledin the art (see ref. 25). Appropriate known methods include cloning theDNA sequence of the wild-type gene into a vector, e.g. a plasmid, andinserting a selectable marker into the cloned DNA sequence or deleting apart of the DNA sequence, resulting in its inactivation. A deletion maybe introduced by, for example, cutting the DNA sequence usingrestriction enzymes that cut at two points in or just outside the codingsequence and ligating together the two ends in the remaining sequence.Alternatively, and more usually now, a mutant allele in which theflanking regions of a target gene are amplified separately and linkeddirectly together in a separate overlap PCR reaction, with omission ofthe intervening target sequence, can be constructed (32). A plasmidcarrying the mutated DNA sequence can be transformed into the bacteriumby known techniques such as electroporation and conjugation. It is thenpossible by suitable selection to identify a mutant wherein theinactivated DNA sequence has recombined into the chromosome of thebacterium and the wild-type DNA sequence has been renderednon-functional by homologous recombination.

Furthermore, the antibiotic resistance genes must generally be removedfrom the bacteria before they are used in a vaccine. Bacteria isolatedfrom the wild often contain antibiotic resistance genes, such asresistance genes against ampicillin, streptomycin, sulphmethoxazole,kanamycin, trimetheprim and tetracyclin. These genes can be removedusing the suicide vector and methods of the invention or by methodsknown to those skilled in the art.

The Nature of the Mutations

The mutations introduced into the bacterial vaccine to preventexpression of enterotoxins or other virulence genes delete or inactivatethe gene. They generally knock-out the function of the gene completely.This may be achieved either by abolishing synthesis of any polypeptideat all from the gene or by making a mutation that results in synthesisof non-functional polypeptide. In order to abolish synthesis ofpolypeptide, either the entire gene or its 5′-end may be deleted. Adeletion or insertion within the coding sequence of a gene may be usedto create a gene that synthesises only non-functional polypeptide (e.g.polypeptide that contains only the N-terminal sequence of the wild-typeprotein). In the case of a toxin gene, the mutation may render the geneproduct non-toxic.

The mutations are generally non-reverting mutations. These are mutationsthat show essentially no reversion back to the wild-type when thebacterium is used as a vaccine. Such mutations include insertions anddeletions. Insertions and deletions are preferably large, typically atleast 10 nucleotides in length up to the length of the entire gene orcoding sequence, for example from 10 to 600 nucleotides. Preferably, thewhole coding sequence or whole gene is deleted.

The mutations are typically site-directed. They may be specific orselective to the toxin gene or other virulence factor gene. In the caseof deleting or inactivating the ST gene in a CFA/I or CS5/CS6 strain,the mutation must specifically target the ST gene without deleting orinactivating the (closely-linked) CFA/I gene, CS5 gene or CS6 gene.

Expression of Heterologous Antigens

An attenuated bacterium of the invention may be genetically engineeredto express an antigen that is not expressed by the native bacterium (a“heterologous antigen”), so that the attenuated bacterium acts as acarrier of the heterologous antigen. In the case that the bacterium isan ETEC bacterium, the antigen may be from another species or fromanother strain of ETEC, so that the vaccine provides protection againstthe other species or strain. Furthermore, the bacterium may beengineered to express more than one heterologous antigen, in which casethe heterologous antigens may be from the same or different species orstrains.

The heterologous antigen may be a complete protein, a part of a proteincontaining an epitope or a fusion protein. The antigen may be fromanother bacterium, a virus, a yeast or a fungus. More especially, theantigenic sequence may be from a pathogenic strain of E. coli (e.g.ETEC). Useful antigens include ETEC colonization factor antigens,non-toxic components or non-toxic mutants of E. coli LT (e.g. the Bsubunit and mutants of the A subunit), LT-ST fusion proteins and choleratoxin B subunit (CT-B) (50-55).

The ETEC CFAs and components thereof are prime candidates for expressionas heterologous antigens. To instigate diarrhoeal disease, pathogenicstrains of ETEC must be able to colonize the intestine and elaborateenterotoxins. For most strains of ETEC, CFAs that are responsible foradhesion to the intestinal mucosa have been identified. In almost allcases CFAs are expressed as fimbriae on the outer surface of thebacteria. A large number of CFAs have been identified, the mostprevalent being CFA/I, CFA/II (includes CS1, CS2, CS3) and CFA/IV(includes CS4, CS5, CS6). Additional antigens include CS17, CS7, CS9,CS14, CS12, PCFO159, PCFO166.

The DNA encoding the heterologous antigen may be expressed from apromoter that is active in vivo. Promoters that have been shown to workwell are the nirB promoter (10, 39), the htrA promoter (10), the pagCpromoter (57) and the ssaH promoter (58). For expression of the ETECcolonization factor antigens or derivatives of LT, CT or ST, thewild-type promoters could be used.

A DNA construct comprising the promoter operably linked to DNA encodingthe heterologous antigen may be made and transformed into the attenuatedbacterium using conventional techniques. Transformants containing theDNA construct may be selected, for example by screening for a selectablemarker on the construct. Bacteria containing the construct may be grownin vitro before being formulated for administration to the host forvaccination purposes.

Plasmid Stabilisation

In order to prevent loss of the plasmid expressing the heterologousantigen or of a native plasmid, an element may be added to the plasmidwhich enhances its stability. It is generally the case that the plasmidsfound in ETEC strains encoding the various colonization factor antigensare low copy number and stable enough to ensure their maintenance overmany generations in the absence of specific selection mechanisms.However, following manipulation of these plasmids in accordance with theinvention, for example to delete genes for toxins such as ST or othervirulence determinants, these stable properties might be impaired. Thisproblem may be alleviated by employing methods for improvement ofplasmid stability.

There are a number of “toxin/antitoxin” plasmid stability determiningsystems known, for example parDE (23) from plasmid RP4 (1), and hok/sok(also known as parB from plasmid R1 or pndAB from plasmid 8483 (11, 12))which could be used to do this. These systems encode two functions:firstly a toxic entity that would kill cells in which it is expressed,which has a long biological half-life, and secondly an antitoxic entitythat prevents this killing but has a short biological half-life. In theevent that a plasmid encoding these functions is segregated duringdivision the daughter cell which does not contain the plasmid exhaustsits supply of antitoxin and is killed by the more persistent toxinmoiety. Thus, only cells that continue to harbour the plasmid aremaintained in the growing population.

Another system that may be used to enhance the stability of a plasmid inaccordance with the invention is a multimer resolution system. Multimerresolution systems confer stability by resolving plasmid multimers intosingle plasmid copies and hence decreasing the chance of plasmid freedaughter cells being generated by random segregation at cell division. Anumber of site-specific recombination systems which act to resolveplasmid multimers into monomers have been identified. In accordance withsuch a system, the plasmid to be stabilised contains a recognition sitefor a site-specific recombinase and the host cell contains a DNAsequence encoding a site-specific recombinase. The recombinase acts onthe recognition site and thereby directs proper segregation of theplasmid during cell division. The recombinase may be encoded on theplasmid to be stabilised or in the chromosome of the host cell.

The recombinase is generally a resolvase. Examples of resolvases whichmay be used in the invention include the Cre recombinase of plasmid P1,the E. coli XerC (ArgR) protein, the D protein recombinase of plasmid F,the ParA recombinases of plasmids RP4 and RK2, the site-specificrecombinase of plasmid R1, resolvases encoded by the Tn3-liketransposable genetic elements and the Rsd resolvase from the Salmonelladublin virulence plasmid.

The recognition elements which may be used in the present inventioninclude those for the above recombinases. Any recognition elementrecognised by the site-specific recombinase employed may be used.Suitable recognition elements include those sites recognised by the XerCsite-specific recombinase, such as the cer site of plasmid ColE1 and thesimilar ckr site of plasmid ColK (59), the psi site of plasmid pSC101and the cer like site of plasmid pHS-2 from Shigella flexneri. Otherrecognition elements which may be used include the crs site from theSalmonella dublin virulence plasmid, the loxP site of plasmid P1, therfs site of the F plasmid and the res site of the Tn3-like transposablegenetic element

In a particularly preferred embodiment of the invention, the recombinaseis the Rsd resolvase which acts via the crs recognition element. TheRsd/crs system is described in detail in our copending application, UKPatent Application No. 0024203.2.

Formulation of Vaccines

The invention provides a vaccine against diarrhoea comprising an E. colicell of the invention and a pharmaceutically acceptable carrier ordiluent. Generally, the vaccine includes a blend of different toxinminus cells (e.g. 2, 3, 4, 5 or 6 cells) which between them carry allthe most common CFAs. For example, the vaccine may contain fivedifferent strains of toxin minus cell as follows:

-   (i) a cell which expresses CFA/I (e.g. ECACC Accession No. 01090303    or 02082967)-   (ii) a cell which expresses CS5 and CS6 (e.g. ECACC Accession No.    01090305 or 02082968);-   (iii) a cell which expresses CS4 and CS6 (e.g. ECACC Accession No.    01090306 or 02082966);-   (iv) a cell which expresses CS2 and CS3 (e.g. ECACC Accession No.    01090304 or 02082964); and-   (v) a cell which expresses CS1 and CS3 (e.g. PTL003, ECACC Accession    No. 01090302 or 02082965).

As mentioned above, the cell deposited under ECACC Accession No.01090302 (PTL003) has already been tested in two clinical trials andbeen shown to be safe and immunogenic.

The vaccine may be formulated using known techniques for formulatingattenuated bacterial vaccines. The vaccine is advantageously presentedfor oral administration, for example as a dried stabilised powder forreconstitution in a suitable buffer prior to administration.Reconstitution is advantageously effected in a buffer at a suitable pHto ensure the viability of the bacteria. In order to protect theattenuated bacteria and the vaccine from gastric acidity, a sodiumbicarbonate preparation is advantageously administered with eachadministration of the vaccine. Alternatively the vaccine is presented ina lyophilised encapsulated form.

The vaccine may be used in the vaccination of a mammalian host,particularly a human host. An infection caused by a microorganism,especially a pathogen, may therefore be prevented by administering aneffective dose of a vaccine prepared according to the invention. Thedosage employed may ultimately be at the discretion of the physician,but will be dependent on various factors including the size and weightof the host and the type of vaccine formulated. However, a dosagecomprising the oral administration of from 10⁷ to 10¹¹, e.g. from 10⁸ to10¹⁰, bacteria per dose may be convenient for a 70 kg adult human host.

EXAMPLES

The Examples described in this section serve to illustrate theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Map of suicide vector plasmid pDM4. u=unknown sequence, unknownlength.

FIG. 2: Map of improved suicide vector pJCB12.

FIG. 3: Diagram of method used to create specific gene deletionconstructs by overlap extension PCR. Step 1=PCR amplification of two DNAfragments. Step 2=overlap extension PCR using DNA products from reaction1 and reaction 2 of step 1 and amplification of the overlap extensionPCR product. R and S stand for restriction enzyme sites.

FIG. 4: Diagram of method used to demonstrate correct integration ofsuicide vector in to targeted locus by linkage PCR.

FIG. 5: Sequence (SEQ ID NO: 1) of the ST gene and flanking regions ofthe plasmid in strain B showing the open reading frame of the structuralgene and the position of all oligonucleotides described in the text anddetailed in Table 1.

FIG. 6: Sequence (SEQ ID NO: 2) of the EAST1 gene and flanking regionsfrom IS1414 (Genbank # AF143819) showing the open reading frame of thestructural gene and the position of all oligonucleotides described inthe text and detailed in Table 1.

FIG. 7: Sequence (SEQ ID NO: 3) of the LT locus showing the position ofthe oligonucleotides used to create the deletion construct. Theunderlined ATG codon near the end of the sequence is the start codon ofLTA.

FIG. 8: Sequence (SEQ ID NO: 4) of the 3′ flanking region of the ST-1gene determined from strain E showing the position of oligonucleotidesdescribed in the text and detailed in Table 1. The open reading frame ofthe structural gene and the ATG and TAA start and stop codons areunderlined.

FIG. 9: Diagram of the individual stages involved in the attenuation ofstrain E.

FIG. 10: Diagram of the individual stages involved in the attenuation ofstrain H.

FIG. 11: Sequence (SEQ ID NO: 5) of the aroC gene locus showing theposition of the oligonucleotides used to create the deletion constructand of others mentioned in the text and described in Table 1.

FIG. 12: Diagram of the individual stages involved in the attenuation ofstrain J.

FIG. 13: Gel showing PCR amplified LT-B coding sequence from strain E.The left hand arrow indicates the LTB PCR product and the right handarrow indicates a 600 bp marker.

FIG. 14: Sequence (SEQ ID NO: 6) of the LT-B gene cloned from strain E.

FIG. 15: Diagram showing construction of plasmid for the expression ofLT-B under control of the nirB promoter. The LTB DNA was digested withBglII and NheI. The starting vector, pNCAT4, was digested with the sameenzymes and the larger vector fragment was isolated. The LTB DNA andvector fragment were ligated together. The KpnI and BglII sites in thefinal plasmid, pNLTB, may be used for cloning of the LTB promoter.

FIG. 16: Western blot showing the expression of LT-B in attenuated ETECvaccine strain PTL003, in both the cytoplasm and periplasm.

FIG. 17: Gel showing PCR amplified LTAB promoter sequence from strain E.The left hand arrow indicates a 200 bp maker and the right hand arrowindicates the LT promoter PCR product.

FIG. 18: Sequence (SEQ ID NO: 7) of LTAB promoter cloned from strain E.

FIG. 19: Western blot showing the expression of LT-B in attenuated ETECvaccine strain PTL003, under control of the native LT promoter. PLLTB-1,-2 and -3 are three independent colonies of PTL003 transformed withpLLTB. The three left had arrows indicate background, the top right handarrow indicates LTA and the bottom right hand arrow indicates LTB.

FIG. 20: Diagram of the “plasmid rescue” technique to obtain thesequence of the 3′ flanking region of ST of strain B. The technique isdescribed in detail in Example 2. An ST cassette was made by PCR(primers 4764 and 4765) and cloned into plasmid pJCB12. The resultingpJCB12-ST plasmid was introduced into strain B by conjugation. PlasmidDNA from one of the transconjugants was isolated and digested with arestriction enzyme (BglII) which cuts at a site distant from the targetgene. The 5′ and 3′ ends of the resulting cut plasmid were ligatedtogether to close the plasmid. The plasmid was propagated in SY327λpirand then sequenced with a primer (4792) which will only hybridise to thenative ST gene (not to the ST gene of pJCB12-ST). The sequence obtainedusing this primer may be used to design further primers to extend thesequence.

EXAMPLE 1: AN IMPROVED PROCESS FOR THE INTRODUCTION OF MULTIPLE GENTICMUTATIONS INTO BACTERIAL VACCINE STRAINS

This section describes the generation of a novel suicide vector plasmid,pJCB12, and its use in the generation of an optimised procedure for theintroduction of mutations into chromosomal or plasmid encoded gene loci.It also describes many of the standard methods used in this andsubsequent examples of the specification, at the end of the section. Thesequence and purpose of the oligonucleotides used in PCR forconstruction or analysis of constructs are given in Table 1 at the endof the Examples.

An Improved Process for the Introduction of Multiple Genetic Mutationsinto Bacterial Vaccine Strains

The generation of an attenuated ETEC strain from a wild-type strainrequires mutation of a number of different genetic loci. These mutationsare introduced sequentially, each using a process that requires severaldifferent steps. When a number of ETEC strains require attenuation, thisamounts to a significant number of steps, each potentially with itsassociated difficulties. It is therefore vital that the process ofintroducing mutations is optimised fully.

Suicide vector plasmids such as pDM4 (20), pJCB12, pCVD442 (8) andothers can be used to introduce defined genetic constructs into specifictargets in the bacterial genome. Plasmid pJCB12 is a new, optimisedsuicide vector based on the previously constructed suicide vector pDM4.The defined genetic construct to be introduced into the bacterial genomemay be a deletion mutation of a specific gene, or a more complexstructure such as, for example, an insertion of a gene within anotherand expressed from a chosen promoter from within the construct.Generally, the extremities of the constructs will consist of nucleotidesequences derived from the region of the genome to be targeted.

Suicide vectors pDM4 and pJCB12 possess a number of key components (seeFIGS. 1 and 2):

An origin of replication which directs replication of the vector in somestrains of bacteria but not in others, oriR6K. oriR6K is the origin ofreplication derived from the naturally occurring plasmid R6K. Thisorigin requires the R6K pir gene for replication, which is absent fromthe suicide vectors. Three laboratory E. coli strains are available thatcarry the pir gene on their chromosome, which are SY327λpir, SM10λpir,and DH5αλpir. All three of these strains may be used to propagate pDM4,pJCB12 and their derivatives.

A transfer origin that directs conjugative transfer of the vector fromone bacterial strain to another, mobRP4. mobRP4 is the transfer originfrom the naturally occurring plasmid RP4. This allows the conjugativetransfer of pDM4 and pJCB12 and their derivatives to recipient bacterialstrains. In order to function, mobRP4 requires the genes encoding theRP4 transfer functions to be present in the donor bacterial cell.Laboratory E. coli strain SM10λpir carries these genes on itschromosome, and so this strain can be used as a donor strain for pDM4,pJCB12 and their derivatives.

A gene encoding a product that is toxic to bacterial cells when thecells are grown under defined conditions, sacB. sacB codes forlevansucrase which produces a product that is toxic to Gram-negativebacteria when grown on sucrose.

A selectable marker, cat. cat codes for chloramphenicolacetyltransferase and confers resistance to the antibacterialchloramphenicol.

A multiple cloning site (MCS), i.e. a site into which defined geneticconstructs may be cloned for introduction into a recipient bacterialcell.

It is clear to us from our use that existing suicide vectors such aspDM4 and pCVD442 frequently does not target the correct locus, andscreening of a large number of transconjugants or transformants isnecessary to identify one that is correctly targeted (if a correctlytargeted one can be identified at all). In addition, pCVD442 carries anampicillin resistance determinant to allow selection of recombinants.However, selection on ampicillin is not as efficient as chloramphenicoland attempts to select for very small numbers of recombinants within avery large mixture of bacteria, such as when performing conjugationexperiments, are often thwarted by “background growth”. “Backgroundgrowth” can take the form of smears of bacteria within whichidentification of transconjugants is not possible, or small colonies,which upon further analysis are not transconjugants.

We believe that the problems of incorrect targeting are partly due tothe relatively large size (6-7 kb) of these known suicide vectors whichare themselves constructed from components of naturally occurringplasmids. The suicide vectors could therefore target naturally occurringplasmids within a bacterial strain that carry similar nucleotidesequences. In particular, transfer functions among conjugative plasmidsare relatively conserved, and the mobRP4 transfer region of pDM4 andpCVD442 is approximately 2.5 kb. In addition, in light of the problem ofincorrect targeting we carried out sequencing. Our nucleotide sequencedata obtained for the sacB region of pDM4 showed that it includesapproximately 600 bp of an IS1-like insertion sequence, an insertionsequence which is prevalent in the genomes of many bacteria. We deducedthat this could be at least partly responsible for the incorrecttargeting.

Suicide vector pJCB12 is a modified version of pDM4 in which much of theintergenic and non-functional DNA has been removed. Therefore, there ismuch less opportunity for incorrect targeting using this suicide vector.Whereas pDM4 is approximately 7 kb in size, pJCB12 is only 3 kb butretains all the key components. In particular, the mobRP4 region ofpJCB12 is merely 0.15 kb, and the IS1-like nucleotide sequences havebeen removed from the sacB region. These modifications are particularlyadvantageous when manipulating ETEC strains which generally harbour manyplasmids that could act as undesirable targets of homologousrecombination with components of the suicide vector. In addition, thesmaller size of to pJCB12 allows easier in vitro manipulation andconstruction of derivatives because smaller DNA molecules ligatetogether and transform into E. coli hosts more efficiently, improvingthe chances of obtaining derivatives of the correct construction. Thesmaller size also allows greater efficiency when introducing theconstructs into recipient bacteria by transformation rather than byconjugation.

Laboratory E. coli strain SM10λpir can be used to transfer pJCB12 andits derivatives to recipient bacterial strains by conjugation because ithas the tra functions from plasmid RP4 inserted into its chromosome.However, strain SM10λpir shows relatively low transformationfrequencies. For this reason, strain DH5αλpir would normally be used forthe construction of pJCB12 derivatives, and once derivatives of thecorrect construction have been identified these would be transferred toSM10λpir for introduction to recipient strains by conjugation.

Construction of Suicide Vector pJCB12

Suicide vector pJCB12 was constructed by several rounds of overlapextension PCR (31, FIG. 3) using pDM4 plasmid DNA as template.Initially, four fragments were amplified from pDM4 by PCR using the highfidelity DNA polymerase, Pfu Turbo™. These were the oriR6K fragment,amplified using oligonucleotides 4714 and 4715; the mobRP4 fragmentamplified using oligonucleotides 4716 and 4717; and the cat gene thatwas amplified in two parts using oligonucleotides 4718 with 4719 and4720 with 4721. This was done in order to remove an EcoRI restrictionenzyme site within the cat gene. The oriR6K fragment and the mobRP4 werethen joined in an overlap extension PCR reaction using oligonucleotides4714 and 4717. Likewise, the cat fragments were joined usingoligonucleotides 4718 and 4721. These two resulting fragments were thenjoined in a final overlap extension PCR reaction using oligonucleotides4717 and 4718. The resulting PCR product was ligated and transformedinto SY327λpir cells and transformants were selected on L-agarsupplemented with chloramphenicol at 20 μg/ml. Transformants harbouringplasmids of the correct size were obtained and one of these, calledpDM4A7, was chosen for further manipulation.

At this stage, clearly the oriR6K and cat components of the plasmidpDM4A7 are functional. However, in order to confirm that the mobRP4locus was functional plasmid pDM4A7 was transformed into strainSM10λpir. These transformants were picked onto L-agar supplemented withchloramphenicol at 15 μg/ml and naladixic acid at 5 μg/ml. This L-agarwas cross-streaked with cells of strain SY327λpir. While chloramphenicolselects those bacterial cells which harbour pDM4A7, nalidixic acidselects for SY327λpir. After overnight incubation, many colonies grewwhere the strains were cross-streaked, but none grew elsewhere on theplate, confirming that pDM4A7 is mobilisable from strain SM10λpir andthat the mobRP4 locus is functional.

Plasmid pDM4A7 was then digested with EcoRI, treated with Pfu Turbo™ DNApolymerase and ligated in order to remove the EcoRI restriction enzymesite to generate plasmid pDM4A7ΔEcoRI. A short HindIII fragment frompDM4 which includes the multiple cloning site was then ligated intopDM4A7ΔEcoRI digested with HindIII. The ligation reaction wastransformed into SY327λpir and transformants selected on L-agarsupplemented with 20 μg/ml chloramphenicol.

Oligonucleotide R6K-01 hybridises within the short HindIII fragment frompDM4 which includes the multiple cloning site. Therefore, transformantswere screened by PCR using oligonucleotides R6K-01 and 4720 in order toidentify those harbouring the desired plasmid construct. A number ofsuch transformants were identified, and one of these, called pDM4A7ΔE,was chosen for further manipulation.

Plasmid pDM4A7ΔE carries three EcoRI sites very close together on theshort HindIII fragment from pDM4 which includes the multiple cloningsite. The two very short EcoRI fragments of pDM4A7ΔE were thereforeremoved by digestion with EcoRI followed by ligation. This resulted in apDM4A7ΔE derivative that possess only one EcoRI site which was calledpJCB10. The region of pJCB10 that includes oriR6K and the MCS wasamplified using oligonucleotides 4715 and 4917 and nucleotide sequencedeterminations for part of this fragment were performed usingoligonucleotide 4917. This presented us with the nucleotide sequenceacross the MCS which was previously unknown.

The sacB gene was then amplified using Pfu DNA polymerase andoligonucleotides 4722 and 4723. The 1.6 kb product was ligated with theplasmid vector pPCR-Script™(Stratagene) and transformed into E. coliXL10 Gold™ cells (Stratagene). Transformants were obtained and thefunctionality of the sacB gene was confirmed by plating the clones ontoL-agar and 5% sucrose agar. One construct gave good growth on L-agar,and none on 5% sucrose agar, and so was chosen as the source of the sacBgene. The sacB gene was then digested from this clone using therestriction enzyme PstI, sites for which were incorporated intooligonucleotides 4722 and 4723 for this purpose, and ligated with pJCB10also digested with PstI. Colonies were checked by PCR usingoligonucleotides 4716 and 4766, yielding a product of the expected size(˜1700 bp). Again the functionality of the gene was confirmed by platingthe clones onto L-agar and 5% sucrose agar. One construct grew onL-agar, but not on 5% sucrose agar. Sequencing of this construct usingoligonucleotides 4716 and 4766 respectively indicated the orientation ofthe sacB gene. This construct was called pJCB12.

Principle of Use of pJCB12

Once a defined genetic construct has been ligated into pJCB12 to give apJCB12-derivative, the plasmid is transferred into a recipient strainsuch as an ETEC strain. This may be done according to methods well knownin the art, either by conjugation from the pJCB12 host strain SM10λpir,or by transformation of the purified pJCB12-derivative directly into therecipient strain.

Transconjugants or transformants are selected on bacteriological growthmedium supplemented with the antibiotic chloramphenicol. Since thesuicide vector pJCB12 is unable to replicate in the absence of the pirgene, any transconjugants or transformants that grow will generally haveresulted from fusion of the pJCB12-derivative with another replicon byhomologous recombination. Using pDM4 and other larger suicide vectorswill often result in incorrect targeting, and consequently mutantscannot be isolated in subsequent time-consuming steps.

Although targeting by pJCB12 is much improved over pDM4, incorrecttargeting can still occur. Therefore, in order to optimise fully thedefined mutation process, a novel approach was taken to screentransformants or transconjugants using PCR to identify those in whichthe pJCB12-derivative has targeted the desired region of the genome. Forthis, one oligonucleotide is designed which hybridises within the pJCB12nucleotide sequences adjacent to the MCS where the defined geneticconstruct has been inserted. The other oligonucleotide is designed tohybridise to the region of the genome to be targeted, adjacent to butoutside of the defined genetic construct. Transformants ortransconjugants that are positive using this PCR will have thepJCB12-derivative targeted to the correct region of the genome (see FIG.4).

Once the correct recombinants have been identified, derivatives need tobe isolated in which the pJCB12 vector has been lost. Such derivativesmay be selected by supplementing the bacteriological growth medium with5% sucrose. This sucrose selection may be made more efficient using amodified L-medium in which the NaCl ingredient is absent andsupplemented with 5% sucrose. Under these conditions the sacB gene ofpJCB12 is toxic, and only derivatives where the sacB gene has been lostwill grow. This event again occurs by homologous recombination and has anumber of outcomes. Firstly, a reversion event will result in thetargeted region remaining as it was. Secondly, homologous recombinationmay result in the defined genetic construct being swapped with thetargeted region resulting in the defined construct being incorporated atthe target region. In addition, if the targeted region is part of aplasmid, such as many of the toxin genes of ETEC strains, then twoadditional events may occur. These are, thirdly, an undefinedspontaneous deletion event, resulting in the loss of a part of thetargeted region which may extend beyond the boundaries of the definedgenetic construct, and, fourthly, the loss of the whole plasmid, anevent which may be termed “specific plasmid curing”.

Testing of sucrose resistant derivatives by PCR can identify the desiredrecombinants. For this, oligonucleotides that hybridise at each end ofthe targeted region and outside of the defined genetic construct areused. If the PCR product is the same size as prior to introduction ofthe pJCB12-derivative construct, then a reversion event has occurred.If, for example the genetically defined construct is a deletionmutation, then the PCR product should be smaller than previously and ofa predictable size. Specific plasmid curing and undefined spontaneousdeletion will normally result in no PCR product or non-specific productsof unexpected size in this type of PCR reaction.

In summary, vector pJCB12 (or another similar vector of the invention)may be used in a method for producing a bacterial cell in which a targetgene (e.g. a toxin gene such as ST, LT or EAST1 or a chromosomal genesuch as an omp or aro gene) is deleted, inactivated or replaced, whichmethod comprises transferring the vector into a bacterial cellcontaining the target gene and selecting for a cell in which the targetgene has been deleted, inactivated or replaced. The selection may becarried out using a multi-stage procedure along the following lines:

-   -   Selecting for a colony of cells which contains the selectable        marker. If the cell into which the vector is transferred is one        that does not support replication of the vector from the origin        of replication in the vector, selecting for such a colony of        cells identifies cells in which the vector has become        incorporated into a cellular replicon;    -   Carrying out PCR to select for a cell in which the vector has        correctly targeted to the target gene, wherein one of the        primers used in the PCR hybridizes to vector sequence adjacent        to the cloning site and the other hybridizes to a site in the        cellular DNA adjacent to the target gene. A positive PCR        indicates that the vector has to targeted to the target gene.    -   Selecting for a cell from which vector sequence has been lost by        growing the cell under conditions which make effective the gene        encoding a product that is toxic to the cells when grown under        defined conditions. Survival of a cell indicates that vector        sequence has been lost. Where the gene encoding the toxic        product is sacB, the cell may be grown in medium supplemented        with sucrose and from which NaCl is absent; the product of sacB        is toxic when the cells are grown in this medium.    -   Finally, PCR may be carried out using primers which hybridize at        positions outside, and adjacent to each end of, the target gene,        wherein a PCR product smaller than the product obtained from a        wild-type cell indicates a deletion mutation.        Materials and Methods

Bacterial strains used. ETEC strains as described elsewhere in thespecification and lab strains of E. coli:

Strain Reference or Source SY327λpir Miller and Mekalanos (56) DH5αλpirP. Barrow, Institute for Animal Health, Compton SM10λpir Simon et al.,1983 (47)

Bacteriological growth media. ETEC strains were routinely grown inL-broth and on L-agar and incubated at 37° C. overnight. L-brothconsists of 10 g/l peptone, 5 g/l yeast extract and 5 g/l of NaCldissolved in 1 l of deionsed water. L-agar is L-broth supplemented with15 g/l agar. Growth medium containing 5% sucrose was as described above,but without the 5 g/l of NaCl. To optimise expression of CFAs ETECstrains were harvested from CFA-agar (1% casamino acids, 2% agar, 0.15%yeast extract, 0.005% MgSO₄, 0.0005% MnCl₂). Chloramphenicol was used ata concentration of 10 μg/ml, tetracycline at 15 μg/ml and streptomycinat 20 μg/ml.

Bacterial Conjugations were performed by mixing donor and recipient ETECstrains on L-agar and incubating at 37° C. for 3 to 18 h. Bacterialgrowth was scraped off into L-broth and plated onto L-agar platessupplemented with chloramphenicol and another appropriate antibiotic toselect ETEC strains (streptomycin for strain B, tetracycline for otherETEC strains) that had incorporated the pJCB12-derivative.

Identification of correctly targeted recombinants. Transconjugants ortransformants obtained by growth on L-agar supplemented withchloramphenicol following introduction of pJCB12-derivative constructswere tested by PCR in order to identify those in which the desiredgenetic locus had been targeted. For this, one of the oligonucleotideshybridised within the pJCB12 nucleotide sequences adjacent to themultiple cloning site (MCS) where the defined genetic construct had beeninserted. The other oligonucleotide hybridised to the genome, adjacentto but outside of the defined genetic construct. In such a PCR, thegeneration of a fragment indicated that the binding sites for therespective oligonucleotides had become linked, which could occur only ifthe pJCB12-derivative had targeted the correct region of the genome.

Excision of pJCB12 from transconjugants by growth in the presence of 5%sucrose. Transconjugants or transformants having the pJCB12-derivativetargeted to the correct region of the genome were then streaked ontofresh L-agar supplemented with chloramphenicol and another appropriateantibiotic to select ETEC strains (see above), and incubated at 37° C.to allow colonies to grow. L-broth cultures inoculated from these freshplates were then grown. Cells from these cultures were harvested,resuspened in 5% sucrose broth, and incubated overnight prior to platingserial dilutions on 5% sucrose agar in order to select recombinants inwhich the pJCB12-derivative had excised. The inoculated sucrose agarplates were then incubated overnight and the resulting colonies testedby PCR using relevant oligonucleotides in order to identify mutants.

DNA manipulations were performed using standard procedures (25). PlasmidDNA was prepared using plasmid purification kits from QIAGEN (Crawley,UK) and DNA fragments were isolated from agarose gels using theQIAquick™ gel extraction kit from QIAGEN.

PCR reactions were performed routinely using Tag DNA polymerase (LifeTechnologies); when high fidelity PCR was required, such as in theconstruction of pJCB12, Pfu “Turbo” DNA polymerase (Stratagene) wasused. Both DNA polymerases were used in accordance with the suppliersinstructions. Routinely, the following PCR cycle was used:

-   Step 1; 95° C., 50 sec-   Step 2; 95° C., 10 sec-   Step 3; 55° C., 1 min-   Step 4; 72° C., 1 min-   Step 5; repeat steps 2 to 4 twenty-five times-   Step 6; 72° C., 1 min

For construction of pJCB12, a pDM4 plasmid DNA preparation was used asthe template in PCR reactions. For routine PCR screening a pick from abacterial colony was used as the template. For construction of toxindeletion mutations, plasmid DNA preparations extracted from suitableETEC strains were used. Plasmid pJCB12 and DNA fragments incorporatingdeletion mutations were generated using a modification of overlapextension PCR (31). In the modified version, there is no overlap in thefragments amplified. Instead, the fragments flank the region to bedeleted, and the complementary sequences which allow joining of the twofragments are included in the 5′-ends of the relevant oligonucleotides;see FIG. 3.

SDS-PAGE

This was performed essentially as described by Laemmli (16) and was usedfor visualisation of expressed CFAs and for checking LPS profiles. ForLPS profiles, cells were grown overnight in L-broth, harvested andresuspended in water so as to give a cell suspension with an A600 of20/cm. The cell suspension was mixed with an equal volume of 50 mMTrisHCl pH6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 0.25% (w/v) bromophenolblue, 2% (v/v) 2-mercaptoethanol and boiled for 5 minutes. Proteinase Kwas then added to a final concentration of 0.2 mg/ml and the samplesincubated at 60° C. for 1 h before loading 5 to 10 μl onto SDS-PAGEgels. For CFAs, bacteria were harvested from CFA-agar and resuspended inwater so as to give a cell suspension with an A600 of 20/cm. The cellsuspension was heated to 65° C. for 10 mins, the samples were thencentrifuged to remove whole cells: An equal volume of 50 mM TrisHClpH6.8, 2% (w/v) SDS, 10% (v/v) glycerol, 0.25% (w/v) bromophenol blue,2% (v/v) 2-mercaptoethanol was then added. Volumes of 3 to 10 μl werethen loaded onto 12% Tris-glycine SDS-PAGE gels (Invitrogen). CFAs werevisualised by Coomassie blue staining (Sambrook et al 1989, ref 25)while LPS was visualised using a SilverXpress™ silver stain kit(Invitrogen).

Example 2 Removal of Toxin Genes and Introduction of AttenuatingMutations into Strains Expressing CFA/I

Manipulation of Strain A to Produce Strain ACAM 2010

Strain A (WS-1858B) expresses the colonisation factor antigen CFA/I andthe heat-stable toxin ST. In order to generate an ST-negative derivativethat continues to express the CFA/I colonisation factors the strain wasfirst transformed with a plasmid conferring tetracycline resistance.This was to allow subsequent selection of the strain from conjugationmixtures. The plasmid conferring tetracycline resistance is a derivativeof plasmid pACYC184 (40) in which the chloramphenicol resistancedeterminant had been inactivated by deletion of a BsmBI restrictionenzyme fragment using standard DNA manipulation procedures. This plasmidwas termed “pACYC-Tc”and was introduced into strain A byelectro-transformation.

The TetR derivative of Strain A was conjugated with SM10λpir harbouringplasmid pJCB12-STI. This pJCB12 derivative contains a fragment of theSTI gene amplified from strain B using oligonucleotides 4764 and 4765cloned into the MCS. The sequence of this construct is given in FIG. 5.Note that the STI fragment in this derivative is not a deletion; the aimis to target the locus with the markers encoded by to pJCB12 in order toallow selection for deletion events resulting in the correct outcome.

Transconjugants were selected by plating the mixture on L-agarsupplemented with tetracycline and chloramphenicol and the coloniesobtained were confirmed as transconjugants by PCR testing usingoligonucleotides 4720 and 4721 which amplifies the chloramphenicolresistance determinant. Three transconjugants were identified and calledA13, A14 and A18 respectively. Each transconjugant was grown on 5%sucrose medium and colonies obtained were screened for ST usingoligonucleotides 4764 and 4765. One of the ST-negative derivativesidentified was called A 18-34 and was tested by PCR for the presence ofthe cfaB gene using oligonucleotides BglIIFOR and BglmodREV, for thepresence of the cfaC gene using oligonucleotides 4727 and 4728, and forthe presence of the cfaR gene using oligonucleotides 4785 and 4786. Allthree of these PCR reactions were positive, confirming the presence ofthe relevant genes. Derivative strain A18-34 was then grown on CFA-agarand processed for SDS-PAGE in order to visualise CFA/I expression. Thisconfirmed that Strain A18-34 expressed CFA/I and, therefore, that anST-negative, CFA/I expressing strain had been isolated.

In the next step an ampicillin sensitive derivative of strain A18-34 wasidentified. For this, strain A18-34 was grown in LB-broth through threepassages. The resulting culture was then diluted and plated ontoLB-agar. When small colonies were visible they were replica-plated ontoLB-agar supplemented with ampicillin at 200 μg/ml and then incubated toallow colonies to grow. One colony was identified that was absent fromthe ampicillin supplemented replica plate and subsequently confirmed asbeing ampicillin sensitive. This strain was called A18-34 Ap^(S).

In the next step, a trimethoprim sensitive derivative of strain A18-34Ap^(S) was isolated. For this strain A18-34 Ap^(S) was replica plated asdescribed above but onto LB-agar and M9 minimal salts agar supplementedwith 0.4% glucose and trimethoprim at 25 μg/ml. Two colonies wereidentified that had not grown on the trimethoprim supplemented replicaplates, and one of these which was called A18-34 Ap^(S) Tp^(S) waschosen for further manipulations.

The EAST1 toxin gene was then deleted from the strain A18-34 Ap^(S)Tp^(s). For this, conjugations were performed with SM10λpir harbouring apJCB12 plasmid derivative carrying a defined EAST1 deletion as the donorstrain and strain A18-34 Ap^(S) Tp^(S) as the recipient strain.Transconjugants were selected on L-agar supplemented with tetracyclineat 15 μg/ml and chloramphenicol at 10 μg/ml. One transconjugant wasidentified by PCR using oligonucleotides 4917 and 4778 in which thepJCB12-derivative was inserted at the correct location. Thistetracycline and chloramphenicol resistant transconjugant was then grownin 5% sucrose medium in order to select recombinants in which thepJCB12-derivative had excised (as described in Materials and Methods).Colonies that grew on L-agar supplemented with 5% sucrose were thentested by PCR using oligonucleotides 4749 and 4752. Three isolates wereidentified that were negative in this PCR reaction indicating that theEAST1 locus had been lost.

Defined deletion mutations were then introduced into the aroC, ompC andompF genes in order to further attenuate the A18-34 Ap^(S) Tp^(S) ΔEAST1derivative as described in other Examples. Initial attempts to introducethe ΔaroC mutation met with little success as was the case whenconstructing a strain J-derivative aroC deletion mutation (see below).Therefore, attempts were made to introduce the ΔaroC^(J) mutation (seebelow for details of the mutation). Following transfer ofpJCB12-ΔaroC^(J) into the A18-34 Ap^(S) Tp^(S) ΔEAST1 derivative atransconjugant was identified by PCR using oligonucleotides 4917 and4742. This transconjugant was then grown in 5% sucrose medium in orderto select recombinants in which the pJCB12-derivative had excised (asdescribed in Materials and Methods). Colonies that grew on L-agarsupplemented with 5% sucrose were then tested by PCR usingoligonucleotides 47116 and 47117, and a derivative that carried theΔaroC^(J) defined deletion mutation was identified. Aromatic amino acidauxotrophy was demonstrated in this A18-34 Ap^(S) Tp^(S) ΔEAST1ΔaroC^(J) strain by streaking on minimal agar and minimal agarsupplemented with “aro mix.” The strain grew only on the aro mixsupplemented agar, whereas the parental strain A 18-34 Ap^(S) Tp^(S)ΔEAST1 grew both in the presence and absence of “aro mix.”

Next the ΔompC defined deletion mutation was introduced into the A18-34Ap^(S) Tp^(S) ΔEAST1 ΔaroC^(J)-derivative as described for strain H(Example 4) and a ΔompC deletion mutant was identified. Expression ofCFA/I and LPS by this A18-34 Ap^(S) Tp^(S) ΔEAST1 ΔaroC^(J) ΔompC strainwas confirmed by SDS-PAGE.

Next the ΔompF defined deletion mutation was introduced into the A18-34Ap^(S) Tp^(S) ΔEAST1 ΔaroC¹ ΔompC-derivative as described for strain H(Example 4) and a ΔompF deletion mutant was identified. Expression ofCFA/I and LPS by this A18-34 Ap^(S) Tp^(S) ΔEAST1 ΔaroC^(J) ΔompC ΔompFstrain was confirmed by SDS-PAGE.

Finally, the pACYC-TC plasmid that was introduced into strain A toconfer tetracycline resistance was specifically cured from the A18-34Ap^(S) Tp^(S) ΔEAST1 ΔaroC^(J) ΔompC ΔompF strain. For this the plasmidpJCB12-pACYCori described in Example 4 was introduced byelectrotransformation. Transformants harbouring pJCB12-pACYCori wereselected by growth on L-agar supplemented with chloramphenicol. Onetransformant was then grown in 5% sucrose medium in order to selectderivatives from which the pJCB12-pACYCori plasmid had been cured (asdescribed in Materials and Methods). Colonies that grew on L-agarsupplemented with 5% sucrose were then picked onto L-agar mediumsupplemented with chloramphenicol and onto L-agar medium supplementedwith tetracyclin. This identified a number of derivatives which weresensitive to both these antibiotics, confirming that the pJCB12-pACYCoriplasmid had indeed specifically cured the strain of the pACYC-Tc markerplasmid and then itself been cured by growth in the presence of sucrose.

One of these antibiotic sensitive derivatives was chosen and tested byPCR for the presence of the cfaA, cfaC, and cfaD genes usingoligonucleotide pairs 47104 with 47105, 4729 with 4730, and 4785 with4786 respectively. In addition, the presence of the ΔaroC^(J), ΔompC andΔompF deletion mutations was confirmed using the oligonucleotide pairs47116 with 47117, 4732 with 4743, and 4733 with TT1. Expression of CFA/Iand LPS was confirmed in this strain using SDS-PAGE and the nucleotidesequence across the ΔaroC^(J), ΔompC and ΔompF mutations was confirmed.

Manipulation of Strain B to Produce Strain ACAM 2011

Strain B (WS-4437A) expresses the colonisation factor antigen CFA/I andthe heat-stable toxin ST. Initially, isolation of a spontaneousST-deletion mutation was attempted by targeting ST using plasmidpJCB12-STI. This plasmid was introduced into Strain B by conjugationfrom SM10λpir and transconjugants were obtained by growth on L-agarsupplemented with chloramphenicol. After several attempts, a number ofST-negative mutants were identified, but these were all negative forCFA/I.

It was therefore decided to construct a defined STI deletion mutationspecific for Strain B. The first requirement was to determine nucleotidesequence flanking the 3′end of the ST gene. The process used to do thisis illustrated in FIG. 20. Plasmid DNA was isolated from atransconjugant strain in which pJCB12-STI was targeted to the ST gene.Purified DNA was subjected to restriction endonuclease digestion withBglII which cuts the ETEC plasmid but not the ST gene or the pJCB12construct. Digested DNA was ligated and electrotransformed intoSY327λpir and transformants were selected on L-agar supplemented withchloramphenicol. This process is termed “plasmid rescue” and results inthe re-isolation of the pJCB12 replicon incorporating a large fragmentof DNA that includes the whole of the STI region and a large amount offlanking DNA. The sequence of the flanking DNA was obtained. Nucleotidesequence data obtained from this then allowed further oligonucleotides(4797 and 4798) to be designed and used to determine additionalnucleotide sequence further downstream of the STI gene in Strain B. FIG.5 shows the determined sequence and the location of all oligonucleotidebinding sites.

Using the determined nucleotide sequence, an ST-deletion mutation wasconstructed. This was done by amplifying two fragments from the STIlocus using oligonucleotides 47101 with 47114, and 47115 with 47100. Thetwo resulting fragments were then joined by overlap extension PCR usingoligonucleotides 47100 and 47101 and ligated into the MCS of pJCB12using standard techniques.

The construct was introduced into strain B by conjugation from SM10λpirand chloramphenicol resistant transconjugants in which ST had beencorrectly targeted were identified by PCR using oligonucleotides 4917and 4799. These transconjugants were grown in the presence of sucroseand colonies obtained were then screened using oligonucleotides 47100and 47101 in order to identify ST deletion mutants. One derivative wasfound which was negative using these oligonucleotides, suggesting that aspontaneous deletion had resulted in loss of the entire ST locus. Thisderivative was positive by PCR using oligonucleotides 4727 with 4728which amplify part of the cfaC gene. It was also positive usingoligonucleotides BglIIFOR and BglmodREV which amplify the cfaA gene, andusing oligonucleotides 4785 and 4786 which amplify the CFA/I regulatorgene, cfaD. Functional assay for ST confirmed that this derivative wasST-negative, while SDS-PAGE confirmed that it continued to expressCFA/I.

Defined deletions were then introduced into the aroC, ompC and ompFgenes in order to attenuate the Strain B ST-negative derivative. Initialattempts to introduce the ΔaroC deletion were unsuccessful. Thereforethe ΔaroC^(J) deletion was used (see below for details of the mutation).Following transfer of the pJCB12ΔaroC^(J) deletion into Strain BST-negative derivative, transconjugants were identified by PCR usingoligonucleotides 4917 with 4742. A transconjugant was identified andthen grown in 5% sucrose medium in order to select recombinants in whichthe pJCB12 derivative had been excised (as described in materials andmethods). Colonies that grew on L-agar supplemented with 5% sucrose werethen tested by PCR using oligonucleotides 47116 with 47117 and aderivative that carried the ΔaroC^(J) deletion was identified. The CFA/Istatus of the exconjugant was checked by PCR using oligonucleotides 4727with 4728 for cfaC, 4785 with 4786 for cfaD and BglIIFOR withBglIImodREV for cfaA.

The ΔompC defined deletion mutation was then introduced into the StrainB ST-negative ΔaroC^(J) derivative as described for strain H (Example 4)and a ΔompC deletion mutant identified. The CFA/I status of theexconjugant was checked by PCR using oligonucleotides 4727 with 4728 forcfaC, 4785 with 4786 for cfaD and to BglIIFOR with BglIImodREV for cfaA.

The ΔompF defined deletion mutation was introduced into the Strain BST-negative ΔaroC^(J) ΔompF ΔompC derivative as described for strain H(Example 4) and an ompF deletion mutant identified. The CFA/I status ofthe exconjugant was checked by PCR using oligonucleotides 4727 with 4728for cfaC, 4785 with 4786 for cfaD and BglIIFOR with BglIImodREV forcfaA.

Finally the pStrep plasmid, that confers streptomycin resistance, wasspecifically cured from the Strain B ST-negative ΔaroC^(J) ΔompF ΔompCderivative. For this a plasmid derivative of pJCB12 was constructedwhich incorporated the pStrep replication origin. This was done byshotgun cloning pStrep fragments generated by restriction endonucleasedigestion with SphI into the SphI site of pJCB12, and transforming theDNA into XL-10 Gold ultracompetent E. coli cells (Stratagene).Transformants were plated on L-agar supplemented with chloramphenicol.Because pJCB12 is a suicide vector requiring special host strains thatcarry the pir gene, transformants that grew in the presence ofchloramphenicol were assumed to be derivatives of pJCB12 which carriedthe pStrep replication origin. Plasmid DNA from four such transformantsshowed that in each case an SphI DNA fragment of approximately 2 kb hadbeen cloned into pJCB12. This pJCB12-derivative was calledpJCB12-pStrep-ORI

The pJCB12-pStrep-ORI plasmid was introduced into the Strain BST-negative ΔaroC^(J) ΔompF ΔompC derivative by conjugation from strainSM10λpir, and the resulting transconjugants selected by growth on agarmedium supplemented with chloramphenicol. Colonies which grew were usedto inoculate broth supplemented with chloramphenicol and, followingincubation to allow growth, dilutions of this culture were plated onagar supplemented with chloramphenicol. Colonies which grew on thismedium were picked onto agar supplemented with streptomycin in order toidentify those from which the pStrep plasmid had been lost. One of thesestreptomycin sensitive colonies was then grown in L-broth supplementedwith 5% sucrose in order to select for a derivative from which thepJCB12-pStrep-ORI plasmid had been lost. Dilutions from this brothculture were plated on L-agar supplemented with 5% sucrose and afterincubation some of the resulting colonies were picked onto agarsupplemented with chloramphenicol in order to identify those from whichthe pJCB12-pStrep-ORI plasmid had been lost.

The CFA/I status of the exconjugant was again checked by PCR usingoligonucleotides 4727 with 4728 for cfaC, 4785 with 4786 for cfaD andBglIIFOR with BglIImodREV for cfaA. CFA/I protein expression was thenchecked by SDS-PAGE and Western blotting. The LPS profile was alsochecked. In addition the ΔaroC^(J), ΔompC and ΔompF mutations wereconfirmed by sequencing, and the aromatic amino acid dependence of thestrain was also confirmed.

Example 3 A Derivative of a Virulent Wild-Type ETEC Strain whichExpresses CS5, CS6, LT, ST and EAST1 wherein the LT, ST and EAST1 Geneshave been Deleted

Strain E expresses CS5, CS6 and the toxins EAST1, ST and LT. In order tofacilitate genetic manipulation plasmid pACYC-Tc (described in Example 2above) was introduced into strain E by electro-transformation in orderto confer tetracycline resistance.

The EAST1 toxin gene was deleted from Strain E first. This required theconstruction of a pJCB12 derivative which carries a defined EAST1deletion mutation. Such a deletion mutation was generated by amplifyingEAST1 fragments from the ETEC strain H10407, using oligonucleotides 4749with 4750, and 4751 with 4752, to generate two DNA fragments flankingthe EAST1 gene. FIG. 6 shows the sequence of the EAST1 locus derivedfrom IS1414 (Genbank accession number AF143819) and all oligonucleotidesused. These were then fused by an additional overlap extension PCRreaction using primers 4749 and 4752 and the resulting fragment wascloned into pJCB12 using the SalI and SphI restriction sites. Thus, apJCB12 derivative had been constructed which incorporated this deletionmutation.

Strain SM10λpir harbouring pJCB12-ΔEAST1 was conjugated with Strain Eand transconjugants selected. Colonies that grew on L-agar supplementedwith chloramphenicol and tetracycline were screened using theoligonucleotides 4917 and 4753 (equivalent to oligos 4 and 1 in FIG. 4).Two transconjugants were obtained which were positive in both this PCRreaction indicating that the pJCB12-ΔEAST1 plasmid had correctlytargeted the EAST1 gene.

These chloramphenicol resistant transconjugants were then grown in 5%sucrose medium in order to select recombinants in which thepJCB12-derivative had excised. Four exconjugants were obtained on thesucrose agar, and these were negative when tested for the presence ofthe EAST1 gene by PCR using oligonucleotides 4749 and 4752, indicatingthat the whole of the EAST1 region had been lost in this derivative.

The strain E EAST1-negative derivative was then tested by PCR usingoligonucleotides 4738 with 4780 which are specific for the CS5 operon,and oligonucleotides 4740 with 4781 which are specific for the CS6operon. These PCR reactions were positive for all four exconjugantsconfirming that they continued to harbour the CS5 and CS6 genes.

Next, an LT-deletion mutant of the Strain E EAST1-negative derivativewas constructed. For this a pJCB12 derivative plasmid was constructedthat carried a defined deletion of the LT-A gene in the LT locus. SeeFIG. 7.

A defined LT-A deletion was constructed by PCR amplification usingoligonucleotides 4772 with 4773, and 4774 with 4746, to generate two DNAfragments flanking the LT-A gene. These were then fused by an additionaloverlap extension PCR reaction and the resulting fragment was clonedinto pJCB12 using the SalI and SphI restriction sites.

The pJCB12-ALT-A construct was introduced into the Strain E ΔEAST1derivative by conjugation from the pJCB12 host strain SM10λpir andtransconjugants were selected on L-agar supplemented withchloramphenicol and tetracycline. Resulting colonies were screened byPCR using oligonucleotides 4762 and R6K-01 which identified twotransconjugants in which the LT locus had been correctly targeted.

These chloramphenicol resistant transconjugants were then grown in 5%sucrose medium in order to select recombinants in which thepJCB12-derivative had been excised. Colonies that grew on the sucroseagar were tested by PCR using oligonucleotides 4762 and 4746 in order toidentify LT-A deletion mutants. Three derivatives were identified whichwere negative in this PCR reaction, suggesting that they had lost theentire LT locus. PCR reactions performed on these three LT-negativederivatives using oligonucleotides 4738 with 4780, and oligonucleotides4740 with 4781 confirmed the presence of the CS5 and CS6 genesrespectively.

Next, the ST gene was deleted from this Strain E EAST1-negativeLT-negative derivative. For this, plasmid pJCB12-STI (as described inExample 2) was transferred into Strain E from SM10λpir andtransconjugant selected on L-agar supplemented with chloramphenicol. Atransconjugant was identified by PCR using oligonucleotides 4917 and4794, and this transconjugant was then grown in 5% sucrose medium toselect for derivatives from which the pJCB12-STI had been lost. Of 133colonies screened, only 3 had lost STI, the others being revertants, andall of these 3 STI-negative derivatives were negative also for CS5 andCS6. These results suggested that the STI locus was present on the sameplasmid as the CS5 and CS6 genes (as did Southern hybridization data)and that the STI locus was relatively stable, so that revertants orderivatives were obtained in which only specific plasmid curing hadoccurred.

It was therefore decided that a defined STI-deletion mutation specificfor strain E was needed. In order to make this construct additionalnucleotide sequence data was required for the region downstream of theSTI gene in Strain E. Therefore, one of the pJCB12-ST transconjugantswas used for sequence determinations at the STI locus using the plasmidrescue procedure described in Example 2 and illustrated in FIG. 20. ApJCB12-derivative was obtained that incorporates a large fragment of DNAthat includes the ST-I gene and a large amount of flanking DNA fromStrain E. This plasmid preparation was used as template in nucleotidesequence determination reactions using oligonucleotide 4764 to determinesequence through the STI gene, and oligonucleotide 4792 to determinesequence downstream of the STI gene. The new sequence data allowed anadditional oligonucleotide, 47106, to be designed which was used infurther nucleotide sequence determinations. This additional new sequencedata enabled oligonucleotides 47112, 47120 and 47121 to be designed forconstruction of the deletion cassette. The sequence of the ST-1 gene andflanking regions showing the binding sites of all oligonucleotides usedis given in FIG. 8.

As with other deletion mutations, two DNA fragments from the ST regionwere amplified by PCR using oligonucleotides 4764 with 47120, and 47121with 47112, and the two fragments generated were fused by an additionaloverlap extension PCR reaction using oligonucleotides 4764 and 47112.The resulting fragment was cloned into pJCB12 using the SacI and SalIrestriction endonuclease sites of pJCB12 and those incorporated intooligonucleotides 4764 and 47112 respectively.

The resulting recombinant plasmid pJBC12-ΔSTI^(E) was introduced intothe Strain E EAST1-negative LT-negative derivative from strain SM10λpir.The resulting chloramphenicol and tetracycline resistant transconjugantswere screened by PCR using oligonucleotides 4917 with 4794, and R6K-01with 47113. Two transconjugants were identified that were positive,indicating that the STI gene had been correctly targeted.

These chloramphenicol resistant transconjugants were then grown inmedium supplemented with sucrose, followed by plating on 5% sucrose agarto obtain excision of the pJCB12 derivative. The colonies that grew onsucrose agar were screened using oligonucleotides 4764 with 47112 and anumber were identified that were negative, and three colonies wereidentified that showed the presence of the STI deletion mutation. PCRreactions performed on the STI deletion mutants using oligonucleotides4738 with 4739 to detect CS5, 4740 and 4741 to detect CS6 and 4783 with4784 to detect the CS5 regulator gene were all positive, indicating thatthese genes had been retained in these defined STI-deletion mutants.Additional attenuating mutations in aroC, ompC and ompF were introducedas described (ref 32 and Example 4). See FIG. 9.

Example 4 Removal of Toxin Genes and Introduction of AttenuatingMutations into Strains Expressing CS2/CS3 (Strain H) and CS4/CS6 (StrainJ)

Strain H Manipulation

Strain H expresses CS2 and CS3 and the toxins EAST and ST. It also hasthe genes for LT, but LT protein has not been detected by in vitroassays. In order to facilitate genetic manipulation the plasmid pACYC-Tc(described in Example 2) was introduced into Strain H byelectro-transformation in order to confer tetracycline resistance.

The EAST1 toxin gene was the first to be deleted from Strain H. StrainSM10λpir containing the pJCB12-ΔEAST1 plasmid described in Example 3 wasconjugated with Strain H and transconjugants selected on L-agarcontaining chloramphenicol and tetracycline. Transconjugant colonies inwhich the EAST1 gene had been correctly targeted were identified by PCRusing the oligonucleotides 4917 and 4753. Transconjugants were thenprocessed in order to select derivatives in which the pJCB12-derivativehad excised (described above). Colonies that grew on 5% sucrose agarwere tested by PCR using the oligonucleotides 4775 and 4777 and a numberwere identified that were negative by PCR indicating that the whole ofthe EAST1 region had been lost. These Strain H EAST1-negativederivatives were then tested for the presence of a transcriptionalactivator for colonization factors, rns, by PCR using theoligonucleotides RNS-03 and RNS-04. Two of the Strain H EAST1-negativemutants were positive for rns. Further testing by PCR for CS2 usingoligonucleotides 4712 and 4779, and CS3 using oligonucleotides CS3-02and CS3-03 indicated that the mutants were both CS2 and CS3 positive.Testing by PCR for the LT locus using oligonucleotides LT-04 and LT-05,and for the STI locus using oligonucleotides EST-01 and 4765 showed thatthe mutants were negative for these toxins, indicating that the ST andLT loci had been lost concomitantly with EAST1. The expression of CS2and CS3 in the Strain H toxin-negative mutants was confirmed bySDS-PAGE.

Defined deletion mutations were then introduced into the aroC, ompC, andompF genes in order to further attenuate the Strain H toxin-negativederivative using a method similar to that described previously (32).However, this earlier description for introducing these mutations usedthe suicide vector pCVD442 described in Example 1. This suicide vectorcodes for ampicillin resistance, which is not optimal for selectinginfrequent transconjugant from a large mixed bacterial population. Inaddition, at this stage, the Strain H toxin-negative derivativecontinues to express it's own ampicillin resistance, making pCVD442useless with this strain. Therefore, the ΔaroC, ΔompC and ΔompF deletionmutations previously cloned into pCVD442 (32) were sub-cloned intopJCB12. For this, the ΔaroC mutation was sub-cloned using therestriction endonuclease sites XbaI and Sad, ΔompC used SacI and SalIsites, and ΔompF used SacI and SphI sites. The pJCB12-derivatives werethen transformed into strain SM10λpir.

The defined ΔaroC mutation was the first to be introduced into theStrain H toxin-derivative from strain SM10λpir. Transconjugant coloniesthat grew on L-agar supplemented with chloramphenicol and tetracyclinewere screened by PCR using the oligonucleotides 4917 and 4742 in orderto identify those in which the aroC locus had been correctly targeted.These transconjugant colonies were then streaked onto fresh L-agarsupplemented with chloramphenicol and tetracycline. Followingincubation, the colonies that grew were tested for the presence rns byPCR using the oligonucleotide primers RNS-03 and RNS-04 and for CS2using primers 4712 and 4779. The reactions confirmed that thetransconjugants were positive for both these loci. Transconjugants werethen processed in order to select derivatives in which thepJCB12-derivative had excised (as described in previous Examples).Colonies that grew on 5% sucrose agar were tested by PCR using theoligonucleotide primers 4731 and TT20 in order to identify derivativesthat harboured the defined ΔaroC deletion mutation. One defined ΔaroCdeletion mutant was identified and again checked by PCR to confirm thepresence of rns and CS2, as described above. Expression of CS2 and CS3by this defined ΔaroC deletion mutant was confirmed using SDS-PAGE, andits LPS was checked using SDS-PAGE.

Next the ompC defined deletion mutation was introduced into this StrainH toxin-negative ΔaroC derivative by conjugation from strain SM10λpirharbouring pJCB12-ΔompC. Transconjugant colonies that grew on L-agarsupplemented with chloramphenicol and tetracycline were screened by PCRusing the oligonucleotides 4917 and 4743 in order to identify those inwhich the ompC locus had been correctly targeted. Transconjugants werethen processed in order to select derivatives in which thepJCB12-derivative had excised (as described above). Colonies that grewon 5% sucrose agar were tested by PCR using the oligonucleotide primers4732 and 4743 in order to identify derivatives that harboured thedefined ΔompC deletion mutation.

The defined ompF mutation was then introduced into this Strain Htoxin-negative ΔaroC ΔompC derivative in a similar fashion to thatdescribed for the ompC mutation above, except that transconjugantcolonies were screened by PCR using the oligonucleotides R6K-01 and 4733in order to identify those in which the ompF locus had been correctlytargeted. Colonies that grew on 5% sucrose agar were tested by PCR usingthe oligonucleotides 4733 and TT1 in order to identify derivatives thatharboured the defined ΔompF deletion mutation.

All of the defined deletion mutations of the Strain H toxin-negativeΔaroC ΔompC ΔompF derivative were then checked by PCR for comparisonwith the wild-type Strain H. For this the oligonucleotides used for aroCwere 4731 and TT20, for ompC were 4732 and 4743, and for ompF were 4733and TT1. The PCR products generated by these reactions were used fornucleotide sequence determinations across the deletion mutations. Theoligonucleotides used for the nucleotide sequence determinationreactions were TT35, TT38 and TT33 for aroC, ompC and ompF respectively.All the deletion mutations had the expected nucleotide sequence. Themutant was also checked by PCR for the presence of rns and CS2, and forthe absence of the toxin loci, as described above. The presence of theCS3 locus was also confirmed by PCR using the oligonucleotides CS3-03and CS3-06. Expression of CS2 and CS3 was confirmed using SDS-PAGE, andthe LPS was checked using SDS-PAGE.

The ampicillin resistance determinant was then removed from the Strain Htoxin-negative ΔaroC, ΔompC, ΔompF derivative. For this the derivativestrain was grown through three passages in L-broth and then dilutionsplated on L-agar in order to obtain well-separated colonies. Thecolonies were then replica plated onto L-agar supplemented withampicillin at 100 μg/ml. After incubation to allow the colonies tore-grow, the L-agar plates were examined in order to identify anycolonies present on the L-agar that did not grow on the ampicillinsupplemented L-agar. One such colony was identified and used in furtherexperiments.

Finally, the pACYC-Tc plasmid that was introduced into Strain H toconfer chloramphenicol resistance was specifically cured from the StrainH toxin-negative ΔaroC, ΔompC, ΔompF derivative. This required theconstruction of the plasmid pJCB12-pACYCori. For this the pACYC-Tcorigin of replication was amplified by PCR using the oligonucleotides4760 and 4761 and cloned into pJCB12 using the restriction endonucleasesites SacI and SalI. Strain SM10λpir containing pJCB12-pACYCori wasconjugated with the Strain H toxin-negative-ΔaroC, ΔompC, ΔompFderivative, and transconjugants were selected on L-agar containingchloramphenicol. It was hoped that by introducing a second plasmidcarrying the same pACYC184 replication origin and selecting for itsmaintenance, the first pACYC-Tc plasmid would be rendered unstable andin the absence of any selection for its maintenance would be morefrequently lost spontaneously. Chloramphenicol resistant transconjugantcolonies harbouring the pJCB12-pACYCori plasmid derivative were pickedonto L-agar supplemented with tetracycline and one of these colonies wasfound to be tetracycline sensitive, indicating that the pACYC-Tc plasmidhad been lost. This tetracycline sensitive, chloramphenicol resistantcolony was then grown on 5% sucrose medium in order to select forderivatives from which the pJCB12-pACYCori had been lost spontaneously.Colonies that grew on 5% sucrose agar were picked onto L-agarsupplemented with chloramphenicol to confirm that the pJCB12-pACYCoriplasmid had indeed been lost.

These manipulations are shown schematically in FIG. 10.

Strain J Manipulation

Strain J expresses CS4 and CS6 and the toxins EAST and ST. It also hasthe genes for LT, but LT protein has not been detected by in vitroassays. In order to facilitate genetic manipulation the plasmid pACYC-Tc(described in Example 2) was introduced into strain J byelectro-transformation thus conferring tetracycline resistance.

The EAST1 toxin gene was deleted from strain J first. Strain SM10λpircontaining pJCB12-ΔEAST1 was conjugated with Strain J andtransconjugants selected on L-agar containing chloramphenicol andtetracycline. Colonies that grew were screened by PCR using theoligonucleotides 4917 and 4753 in order to identify those in which theEAST1 gene had been correctly targeted. These transconjugant colonieswere then streaked onto fresh L-agar supplemented with chloramphenicoland tetracycline and incubated to allow colonies to grow. These colonieswere tested by PCR to confirm the continued presence of the CS4 operonusing the oligonuncleotides 4768 and 4769, and for the CS6 operon usingoligonuncleotide 4740 and 4781.

One transconjugant which was positive for both these PCR reactions wasprocessed in order to select derivatives in which the pJCB12-derivativeplasmid had excised (described above). Colonies that grew on 5% sucroseagar were tested by PCR using the oligonucleotides 4749 and 4752 toidentify EAST1 mutants. All colonies tested were negative by this PCRreaction indicating that the whole of the EAST1 region had been lost inthese derivatives. Further PCR reactions were performed to test for thecontinued presence of the CS4 and CS6 genes as described above, andexpression of CS6 was confirmed by SDS-PAGE.

PCR reactions using oligonucleotides LT-04 and LT-05 amplified a productof the expected size for the LT locus in the Strain J EAST1-negativederivative. Nucleotide sequence determination reactions using these sameoligonucleotides showed that this fragment generated by PCR was indeedof the LT locus. Therefore, this locus was targeted using thepJCB12-ΔLT-A construct in the same way as described in Example 3.LT-negative derivatives were identified in which the whole of the LTlocus had been deleted. Further PCR reactions to test for the presenceof CS4 using oligonucleotides 4768 and 4769, and CS6 using theoligonucleotides 4740 and 4781 confirmed the continued presence of theseloci in the LT-negative derivatives. The to expression of CS6 by theStrain J EAST1-negative LT-negative derivatives was confirmed bySDS-PAGE.

PCR reactions performed using the oligonucleotides ST-01 and ST-02indicated that the ST present in Strain J was that encoded by thetransposon Tn1681 (41). The Strain J EAST1-negative LT-negativederivatives were tested by PCR using the oligonucleotides ST-01 andST-02, and were shown to be ST-negative.

The aroC locus of the Strain J toxin-negative derivative was targetedusing pJCB12-ΔaroC as described above for Strain H. Correctly targetedtransconjugants were identified. Unusually, however, following growth on5% sucrose medium all derivatives generated in a large number ofexperiments were identified as revertants. Therefore a new pJCB12-ΔaroCconstruct was made that incorporated a smaller deletion in the aroClocus. This was constructed by PCR amplification using oligonucleotides47116 with 47118, and 47119 with 47117, to generate two DNA fragmentsflanking that region of the aroC gene to be deleted. These were thenfused by an additional overlap extension PCR reaction usingoligonucleotides 47116 with 47117, and the resulting fragment was clonedinto pJCB12 using the XbaI and Sad restriction endonuclease sites. Thisconstruct was called pJCB12-ΔaroC^(J) and was electrotransformed intoSM10λpir. The sequence of the aroC gene and the binding sites of theoligonucleotides used to construct this novel deletion construct areshown in FIG. 11.

While pJCB12-ΔaroC^(J) was undergoing construction, the ompC defineddeletion mutation was introduced into the Strain J toxin-negativederivative using the pJCB12-ΔompC construct and procedure described forStrain H. One ompC defined deletion mutant was identified by PCR usingoligonucleotides 4732 and 4743.

The defined aroC deletion mutation was then introduced into this StrainJ toxin-negative ΔompC mutant using the new pJCB12-ΔaroC^(J) constructby conjugation from strain SM10λpir. Colonies that grew on L-agarsupplemented with chloramphenicol and tetracycline were screened by PCRusing the oligonucleotides 4917 and 4742 in order to identify those inwhich the aroC gene had been correctly targeted. Transconjugants werethen processed in order to select derivatives in which thepJCB12-derivative plasmid had excised (described above). Colonies thatgrew on 5% sucrose agar were tested by PCR using the oligonucleotides4731 and TT20 in order to identify those with the incorporated definedaroC^(J) deletion mutation.

The defined ompF deletion mutation was then incorporated into anidentified Strain J toxin-negative ΔompC ΔaroC derivative. This was doneexactly as described above for strain H, and one ompF deletion mutantwas identified.

The tetracycline resistant plasmid pACYC-Tc was then specifically curedfrom the toxin-negative ΔompC ΔaroC ΔompF derivative of Strain J usingpJCB12-pACYCori and then this plasmid itself was removed, again asdescribed above for Strain H.

Finally, PCR reactions were performed to confirm the presence of CS4using oligonucleotides 4768 with 4769, and CS6 using theoligonucleotides 4740 with 4781. Similarly the presence of the CFA/IVregulatory gene was confirmed using oligonucleotides 4785 and 4786. Thenucleotide sequences of the defined aroC, ompC and ompF deletionmutations were determined as described for Strain H, and were asexpected. The absence of EAST1, LT and ST loci were again confirmed byPCR as described above, expression of CS6 was confirmed by SDS-PAGE, andthe LPS also was checked using SDS-PAGE.

A diagram showing all of the steps involved in these manipulations isshown in FIG. 12.

Example 5 Increasing the Stability of Manipulated CFA Plasmids

This Example describes as an illustration one system for stabilising aCFA plasmid. The parDE locus was used as the stabilizing moiety.

In order to obtain a derivative of an ETEC strain that does not code forST but continues to express its CFA genes, a pJCB12-ΔSTI derivative isconstructed where the parDE locus is flanked by regions homologous tothe regions flanking the ST gene in the plasmid to be targeted.Introduction of this plasmid into a recipient strain and selection onchloramphenicol will now result in transconjugants in which both lociare present in the CFA plasmid. The functional parDE locus will nowensure that this plasmid is maintained in these cells. Growth on sucroseto identify derivatives from which the pJCB12 component has been lostwill now strongly select for cells containing a recombinant plasmid inwhich the desired cross over (ie the replacement of the ST structuralgene with the parDE locus) has taken place. Cells in which a reversionevent has occurred or from which the entire plasmid has been lost willbe killed by the action of the toxin. Incorporation of the parDE locuswill also function to stabilise the inheritance of the CFA plasmidduring subsequent rounds of mutation, for example the introduction ofadditional attenuating mutations such as in the aroC, ompC and ompFgenes.

Oligonucleotides 4789 and 4790 are used to amplify the parDE locus fromplasmid RP4. The purified PCR product is then cloned into pJCB12-ΔSTIusing the XhoI restriction enzyme sites to give plasmidpJCB12-ΔSTI:parDE. This plasmid is now introduced into a recipientstrain by conjugation from SM10λpir or electro-transformation withpurified plasmid DNA and the required intermediates and derivativesisolated by methods described in detail in the previous Examples.

Example 6 Expression of LT-B in an Attenuated ETEC Strain, PTL003, toInduce a Protective Immune Response Against LT

Aim

The aim of this work was to express the B-subunit of Escherichia coliheat labile toxin in a vaccine strain of ETEC. The LTB gene was derivedfrom strain WS2773-E (NAMRU3, Cairo, Egypt) but it could be derived fromany LTB-encoding strain. Similarly the approach could be extended toinclude mutants of LT-A, LT-B fused to other proteins (eg ST) or toexpression of CT-B or derivatives thereof. Initial plasmid constructswere designed to demonstrate that LT-B could be expressed in an ETECvaccine strain in the absence of LT-A and could be correctly exportedand assembled. Subsequent constructs used the native LT promoter todrive expression. Ultimately, a similar construct could be inserted intothe chromosome of ETEC to create a stable strain without the need forantibiotic selection.

Methods

Section 1—PCR Amplification of the LTB CDS (Protein Coding Sequence)

Primers

Genbank sequences were used to design appropriate PCR primers (Table 1).The forward primer (Bfor) was designed to amplify from the start codonof the LTB gene and was based on Genbank sequence M17874 (17). Tofacilitate cloning into expression vectors, a BglII restriction site wasincluded starting 8 bases 5′ prime of the ATG start codon. The reverseprimer (Brev) was designed to amplify from 200 bases downstream of thestop codon, such that any transcription terminators would be included inthe PCR product, and was based on Genbank sequence AF190919 (26). AnNheI restriction site was included in the primer to facilitate cloninginto expression vectors.

Template

Plasmid DNA was isolated from Strain WS2773-E (NAMRU3, Cairo, Egypt) foruse as a template.

Reaction

Reaction mixture 2 μl plasmid DNA from WS2773-E (approx 50 ng/μl) 10 μl10X Pfu DNA Polymerase buffer (Stratagene ™) 0.8 μl dNTPs (25 mM of eachdNTP) 0.5 μl primer BFOR (258 ng/μl) 0.5 μl primer BREV (227 ng/μl) 84μl H₂O 2 μl PfuTurbo ™ DNA Polymerase(2.5 U/μl) Program  1 cycle 94° C.× 1 min 30 cycles 94° C. × 1 min 56° C. × 1 min 72° C. × 1 min  1 cycle72° C. × 10 min Hold  4° C.Results

A 600 bp PCR product was synthesized and isolated from a 1% agarose gelusing a QIAquick™ gel extraction kit (Qiagen) according to themanufacturer's instructions (FIG. 13).

Section 2—Cloning of the LTB PCR Product

Cloning into pPCR-Script Amp SK+

Gel isolated PCR product was ligated into pPCR-Script Amp SK+™(Stratagene) according to the instructions in the manufacturer'sinstruction manual (#211188). 2 μl of ligation mix was used to transformE. coli XL10-Gold™ Supercompetent cells (Stratagene #230350) and correctconstructs were identified by digestion of purified plasmid DNA withPvuII. A correct construct was designated pPCRLTB. The LTB gene wasfully sequenced (FIG. 14) and was compared to Genbank sequence M17874(17). Four base changes were found which resulted in two amino acidchanges. The PCR was repeated and a second clone was sequenced and gaveidentical results, showing that the LT-B sequence in strain WS2773-Ediffers from the database sequence.

Cloning into an Inducible Expression Vector

The LTB gene was transferred into expression vector pNCAT4 (FIG. 15).pNCAT4 is an expression vector designed for use in Salmonellas typhi andtyphimurium. The vector was originally derived from pTETnir15 (3) buthad been modified by replacement of the TetC gene by the H. pyloricatalase gene and replacement of the bla ampicillin resistance gene by akanamycin resistance gene. In this plasmid the catalase gene is underthe control of the nirB promoter which is upregulated in anaerobicconditions. For the purpose of the present invention many alternativeexpression plasmids which are well known in the art could be substitutedfor pNCAT4 or pTETnir 15.

Plasmid pPCRLTB was digested with restriction enzymes BglII and NheI anda 600 bp fragment containing the LTB gene was isolated from a 1% agarosegel using a QIAquick™ gel extraction kit (Qiagen) according to themanufacturer's instructions. Plasmid pNCAT4 was digested withrestriction enzymes BglII and NheI and a 2.6 kb vector fragment wasisolated from a 1% agarose gel using a QIAquick™ gel extraction kit(Qiagen) according to the manufacturer's instructions.

The vector was ligated to the LTB gene (25) and the ligation mixture wasused to transform E. coli XL10-Gold™ supercompetent cells (Stratagene#230350). Correct clones were identified by restriction enzyme analysisand one (designated pNLTB) was used to transform electrocompetentETEC-PTL003.

Section 3—Expression and Localization of LTB in ETEC-PTL003

Sample Preparation

Cultures of PTL003 and PTL003-pNLTB were grown overnight in LB mediumwith appropriate antibiotics. Samples (1 ml) were diluted in 100 mlmedium and grown with shaking for ˜3 h. Flasks were transferred to astatic incubator and growth was continued for a further 1.5 h.

Bacteria were fractionated into periplasmic and spheroplast componentsby the following method: Cells were harvested by centrifugation for 5min at 10 400×g. The cell pellet was resuspended in 5 ml of 200 mM TrisHCl, pH8.0 and was transferred to a 50 ml glass beaker containing amagnetic flea. To this was added 5 ml of TES buffer (200 mM Tris HCl, pH8.0, 1 mM Na₂EDTA, 1 M sucrose). Stirring was set in motion and a timerwas started. At t=90 s, 1 mg of lysozyme (10 mg/ml solution) was added,and at t=135s, 10 ml of water was added; the mixture was incubated atroom temperature for a further 30 min. Spheroplast formation was checkedwith a light microscope and, if complete, the preparation wascentrifuged for 20 min at 47 500×g at 20° C. The supernatant containingthe periplasmic proteins was harvested and concentrated˜two-fold in aCentricon Macrosep™ spin-concentrator (3 000 NMWL, Millipore). Thepellet, containing spheroplast proteins (cytoplasmic andmembrane-bound), was resuspended in PBS. Samples of the cell fractionswere mixed with equal quantities of Tris-Glycine SDS-PAGE sample buffer(Invitrogen LC267) containing 0.1 M dithiothreitol. A portion of theperiplasmic sample was heated at 95° C. for 5 min, the remainder waskept at room temperature.

Sample Analysis

Samples were separated by electrophoresis on 18% Tris-Glycine Gels(Invitrogen EC6505) and transferred onto nitrocellulose membranesaccording to the instructions supplied with the Xcell II Blot Module™(Invitrogen EI9051). Blots were incubated as follows:

Block 1 hour in PBS, 0.05% Tween 20 ™ containing 5% dried milk(Marvel ™) with gentle rocking. Primary 1 hour with rabbit anti-choleratoxin antibody (Sigma C3062) antibody diluted 1:10 000 in PBS, 0.05%Tween 20 ™ containing 1% Marvel and a 1:2 000 dilution of an E. coliextract (Promega #S3761). The antibody/extract mixture was pre-incubatedfor 1 hour before use to reduce non-specific binding to E. coliproteins. Wash Three 5 min washes in PBS, Tween 20 ™, 1% Marvel ™.Secondary 1 hour with horseradish peroxidase conjugated anti-rabbitantibody antibody (Sigma A4914) diluted 1:10 000 in PBS, 0.05% Tween20 ™, 1% Marvel ™. Wash Three 5 min washes in PBS, Tween 20 ™, 1%Marvel ™. Two 5 min washes in PBS, Tween 20 ™ Two 5 min washes in PBSDevelop Blots were developed using a SuperSignal West PicoChemiluminescent Substrate ™ kit (Pierce).

The results of the Western blots are presented in FIG. 16. LTB monomerswere detected in both spheroplast and periplasmic boiled fractions. Inthe periplasmic samples kept at room temperature LTB pentamers weredetected indicating that the LTB could be transported across the cellmembrane and assembled in the normal way.

Section 4—PCR Amplification of the LTB Promoter

Primers

PCR primers were designed based on preliminary sequence data of theupstream region of the LTA gene of WS2773-E (FIG. 7). The forward primer(Pfor) annealed˜200 bp upstream of the LTA gene. A KpnI restriction sitewas included in the primer to facilitate cloning into expressionvectors. The reverse primer (Prev) annealed just upstream of the startcodon of the LTA gene and was designed to introduce a BglII restrictionsite to allow correct positioning of the promoter fragment with respectto the LTB gene.

Template

Plasmid DNA was isolated from Strain WS2773-E (NAMRU3, Cairo, Egypt) foruse as a template.

Reaction

Reaction conditions were as described for the LTB gene in Section 1.

Results

A 200 bp PCR product was synthesized and isolated from a 1% agarose gelusing a QIAquick™ gel extraction kit (Qiagen) according to themanufacturer's instructions. (FIG. 17)

Section 5—Cloning of the LT Promoter

Cloning into pPCR-Script Amp SK+

Gel isolated PCR product was ligated into pPCR-Script Amp SK™+(Stratagene) according to the instructions in the manufacturer'sinstruction manual (#211188). 2 μl of ligation mix was used to transformE. coli XL10-Gold™ Supercompetent cells (Stratagene #230350) and correctconstructs were identified by digestion of purified plasmid DNA withPvuII. A correct construct was designated pPCRProm. The sequence of thisconstruct is described in FIG. 18.

Cloning into an LTB Expression Vector

Plasmid pPCRProm was digested with KpnI and BglII and the 200 bppromoter fragment was isolated from a 1% agarose gel using a QIAquick™gel extraction kit (Qiagen) according to the manufacturer'sinstructions. The nirB promoter of pNLTB was excised by digested withKpnI and BglII (FIG. 15) and the remaining 3.7 kb vector fragment wasisolated from a 1% agarose gel using a QIAquick™ gel extraction kit(Qiagen) according to the manufacturer's instructions. The vector wasligated to the LT promoter fragment (Sambrook et al., 1989, ref 13) andthe ligation mixture was used to transform E. coli XL10-Gold™supercompetent cells (Stratagene #230350). Correct clones wereidentified by restriction enzyme analysis of purified plasmid DNA andone (designated pLLTB) was used to transform electrocompetentETEC-PTL003.

Section 6—Expression of LTB Under the Control of the LT Promoter

Sample Preparation

Cultures of PTL003 and PTL003-pLLTB were grown overnight in LB mediumwith appropriate antibiotics. Absorbance at 600 nm was used to determinethe concentration of cells in the cultures. Aliquots containing 5×10⁷cells were concentrated by centrifugation and the pellets wereresuspended in 10 μl Tris-Glycine SDS-PAGE sample buffer (InvitrogenLC267) containing 0.1 M dithiothreitol.

Sample Analysis

Samples were analysed by SDS-PAGE (16) and Western blotting exactly asdescribed in Section 3.

Results are presented in FIG. 19. LTB was expressed in PTL003 under thecontrol of the LTAB promoter.

TABLE 1 Oligonucleotides used Name Nucleotide sequence Target locus; use 4712 5′-GTAACTGCTAGCGTTGATCC cotA; detection of CS2 locus  47145′-TTCAACCTTAAAAGCTTTAAAAGCCT oriR6K; construction of pJCB12  47155′-CTACACGAACTCTGAAGATCAGCAGTTCAACC oriR6K; construction of pJCB12  47165′-GATCTTCAGAGTTCGTGTAGACTTTCCTTGG mobRP4; construction of pJCB12  47175′-GCCACTGCAGCCTCGCAGAGCAGGATTC mobRP4; construction of pJCB12  47185′-GGCACTGCAGGCGTAGCACCAGGCGTTT cat; construction of pJCB12  47195′-TCATCCGGAGTTCCGTATGGCAAT cat; construction of pJCB12  47205′-TGCCATACGGAACTCCGGATGAG cat; construction of pJCB12  47215′-GCTTTTAAAGCTTTTAAGGTTGAATTC cat; construction of pJCB12GATCGGCACGTAAGAGGTTC  4722 5′-GGCCTGCAGGCAAGACCTAAAATGTGsacB; construction of pJCB12  4723 5′-GCGCTGCAGCTTTATGTTGATAAGAAAsacB; construction of pJCB12  4727 5′-GCCGCATGCATTAATTCCATATATAGGGGcfaC; detection of CFA/I  4728 5′-GCCGTCGACTGCCATAAGGTAAACGAGCcfaC; detection of CFA/I  4731 5′-GAATTTTACGTCGATGAACGCGaroC; confirmation of linkage with pJCB12 and mutation  47325′-GTACAAATAACCTACAAAAAGCCC ompC  4733 5′-ACCCACACACGCTTAACGCTGG ompF 4738 5′-GGAAAGAGAGTATATCTATGTAACGC detection of CS5  47395′-CGGTCGAGTAATAAGCTGTACTCTGC detection of CS5  47405′-TAATTCTTGCTTCATTCGGCAGCC cssA; detection of CS6  47415′-TAGTAACCAACCATAACCTGATCG cssA; detection of CS6  47425′-CTTCACACTCCAGACTATCGGC aroC; confirmation of mutation  47435′-TTCCTGGCTCGGAATTTGAACC ompC  4746 5′-CGGCATGCCGCAATTGAATTGGGGGeltB; construction of ΔLT-A  4748 5′-AGAACTGCTGGGTATGTGGCTGGEAST1; confirmation of linkage with pJCB12  47495′-GGCGTCGACGAAAATGAAGGGGCGAAGTTCEAST1; construction of EAST1 deletion mutation  47505′-ATGACACGAATGTTGATGGCATCCGGGAAGCEAST1; construction of EAST1 deletion mutation  47515′-GCCATCAACATTCGTGTCATGGAAGGACTACEAST1; construction of EAST1 deletion mutation  47525′-GGCGCATGCAAGATTCGGCCAGTTAGCCEASTI; construction of EAST1 deletion mutation  47535′-GTTGGATAAGCGAAGAACGTGG EAST1; to check for linkage with pJCB12  47605′-GCGGTCGACTGCGGCGAGCGGAAATGGCori pACYC184; construction of pJCB12-pACYCori  47615′-GCCGAATTCAACTTATATCGTATGGGGCori pACYC184; construction of pJCB12-pACYCori  47625′-GGAAGTTGCGTCCATTTTACGGG LT, construction of ΔLT-A  47645′-AATATTACTATGCTCTTCGTAGCGGST strain A; construction of ST deletion mutation  47655′-ATTAATAGCACCCGGTACAAGCAGGST strain A; construction of ST deletion mutation  47665′-CAACAGTACTGCGATGAGTGGcat; nucleotide sequence determinations into sacB  47685′-CAATTGATATTTTGCAAGCTGATGG csfA; detection of CS4  47695′-TAGAAACGACCCCACTATAATTTCC csfA; detection of CS4  47725′-CCGTCGACTAAAAATCACCACCACTTC LT, construction of ΔLT-A  47735′-ATTCATCCTCCTTATATATCATACAAGAAGACAATCC LT, construction of ΔLT-A  47745′-GATATATAAGGAGGATGAATTAT LT, construction of ΔLT-A GAATAAAGTAAAATTT 4775 5′-GTTCAATCCAGCATCAAATGAAG detection of EAST1  47775′-GCCGCATGCCATTCGCCAGTCCTTCAA detection of EAST1  47785′-CCAGGCGGTCACCGAACTCG  4779 5′-TTGAACAGAAAGAAAACTCGCACCcotC; detection of CS2  4780 5′-ATGAATTCTCTCCAACGCTCTTCCdetection of CS5  4781 5′-AGTCAAATGTCCTGCATAAGTACCcssB; detection of CS6  4783 5′-GGATATATCTTTTGGTGAAGATAAGcsvR; detection of CS5 regulator gene  4784 5′-AATAAGATGCGCTAGAAATCCCcsvR; detection of CS5 regulator gene  4785 5′-TATGGATATATATTCAGAAGAAGAGcfaD; detection of CFA/I regulatory gene.  47865′-AATAAGACGCACTGGAAATTCC cfaD; detection of CFA/I regulatory gene  47895′-GGCCTCGAGATTTTCCCGACCTTAATGCGparDE; construction of pJCB12-ΔSTI^(B)::parDE  47905′-CGGCTCGAGGACGTTGTGAGTGGCGCGparDE; construction of pJCB12-ΔSTI^(B)::parDE  47925′-GTGCTATTAATAATATAAAGGGSTI^(B), nucleotide sequencing downstream of STI in StrainB  47945′-TTTTCGGTCGCCGAAAAAGATAATASTI,^(B)sequencing upstream of STI and confirmation of linkage  47975′-GCGCTGTTCTTCAACTGTGG STI^(B), nucleotide sequencing downstream of STIin StrainB  4798 5′-CCACAGTTGAAGAACAGCGCSTI^(B), nucleotide sequencing downstream of STI in StrainB  47995′-ATGTCGCCACGCATGACGGC STI^(B), construction of pJCB12-ΔSTI^(B) 471005′-CCGGCATGCGATGCCCTGCAGATGG STI^(B), construction of pJCB12-ΔSTI^(B)47101 5′-GCCGTCGACTATGCTCTTCGTAGCGGAGSTI^(B), construction of pJCB12-ΔSTI^(B) 471065′-GAACTTTTGCTGAGTTGAAGGAGCSTI^(E), nucleotide sequencing downstream of STI in Strain E 471125′-GGTCAGCCGGAATACGCGTT STI^(E), construction of pJCB12-ΔSTI^(E) 471135′-TCAGGCACAGCTAGCCGTCTSTI^(E), confirmation of linkage of STI in Strain E 471145′-ACAGCGCCTCGAGACTATTCATGCTTTCAGGACCSTI^(B), construction of pJCB12-ΔSTI^(B) 471155′-GAATAGTCTCGAGGCGCTGTTCTTCAACTGTGGSTI^(B), construction of pJCB12-ΔSTI^(B) 471165′-GCGTCTAGACACAACAATAACGGAGCCGTG aroC; construction of pJCB12-ΔaroC^(J)47117 5′-GGCGAGCTCGGAATATCAGTCTTCACATCGGaroC; construction of pJCB12-ΔaroC^(J) 471185′-CCACGCCTTTCACCCCACCGCCGCGATAATCGCaroC; construction of pJCB12-ΔaroC^(J) 471195′-CGCGGCGGTGGGGTGAAAGGCGTGGAAATTGGCaroC; construction of pJCB12-ΔaroC^(J) 471205′-CATCAGAATCACTATTCATGCTTTCAGGACCACSTI^(E), construction of pJCB12-ΔSTI^(E) 471215′-CATGAATAGTGATTCTGATGATGTCTGTAACGSTI^(E), construction of pJCB12-ΔSTI^(E)  4917 5′-ATCAACGGTGGTATATCCAGTcat of pJCB12; confirmation of linkage. Bfor5′-ACGTAGATCTTTATGAATAAAGTAAAATTTTATG LT-B; BglIIFOR5′-CCCAGATCTATATGCATAAATTATTCTATTTACTAAG cfaB; detection of CFA/IBgIIImodREV 5′-CACTTGGTAAAGACCTAATTAGAGCCGC cfaB; detection of CFA/IBrev 5′-GTACGCTAGCCATGTATCTCATTAGCTG LT-B; CS3-025′-TTGTCGAAGTAATTGTTATA detection of CS3 genes CS3-035′-GTGAATGTATGAGGGATTCGA detection of CS3 genes CS3-065′-CTAAATGTTCGTTACCTTCAGTGG detection of CS3 genes EST-015′-CATGTTCCGGAGGTAATATGAA detection of ST gene LT-045′-CATCGCCATTATATGCAAATGGCG eltA; detection of LT genes LT-055′-ACTGATTGCCGCAATTGAATTGGG eltB; detection of LT genes Pfor5′-CCGGTACCATGATTCAATGTACACC LT promoter Prev5′-ACGTAGATCTACTTATATATCATACAAG LT promoter R6K-015′-GTGACACAGGAACACTTAACGGC oriR6K; confirmation of linkage RNS-035′-ACATCATAGCGATGGCATCAA rns; detection of CFA/II regulatory gene RNS-045′-TATTTCAATTCAGTTCGCATCGC rns; detection of CFA/II regulatory geneST-01 5′-CATGACGGGAGGTAACATGA Detection of ST_(Tn1681) ST-025′-TATGCTTTTTAATAACATCC Detection of ST_(Tn1681) TT15′-ATCTGTTTGTTGAGCTCAGCAATCTATTTGCAACC ompF TT205′-ATGCGCGCGAGAGCTCAACCAGGGTCGCACTTTG aroC TT33 5′-TTGTAGCACTTTCACGGTAGompF; nucleotide sequence determinations across ΔompF TT355′-GATGGTGTGTTTATGCTC aroC; nucleotide sequence determinationsacross ΔaroC TT38 5′-GGAGAATGGACTTGCCGACompC; nucleotide sequence determinations across ΔompC 471045′-TTATTGATGGAAGCTCAGGAGG 47105 5′-TAACGCCTGCTCTAACATTCCC  47925′-GTGCTATTAATAATATAAAGGG  4730 5′-TTCTTCACGAACTAATTGAGTG

TABLE 2 GenBank Accession numbers for sequence data EAST1 (astA)AF143819 ST (estA) M18346 LT-A (eltA) V00275 LT-B (eltB) M17874 CFA/Ioperon M55661 CS2 operon Z47800 CS3 operon X16944 CS4 operon AF296132CS5 operon AJ224079 CS6 operon U04844 cfaD M55609 csvR X60106 rns J04166parDE RK2 L05507 sacB X02730 oriR6K M65025 mobRP4 X54459 cat V00622References

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1. A composition which: (a) induces an immune response to colonizationfactor CFA/I, and comprises a bacterial cell which expressescolonization factor antigen CFA/I from a native plasmid but does notexpress heat stable toxin (ST)or (b) induces an immune response tocolonization factor CS5 and/or colonization factor CS6, and comprises abacterial cell which expresses colonization factor antigen CS5 from anative plasmid and/or expresses colonization factor antigen CS6 from anative plasmid but does not express heat stable toxin (ST); wherein thecell is attenuated by deletion or inactivation of one or more gene(s)selected from the group consisting of aroA, aroC, aroD, aroE, pur, htrA,ompC, ompF, ompR, cya, crp, phoP, surA, rfaY, dksA, hupA, sipC and cIpB.2. A composition according to claim 1, wherein said bacterial cell is anEscherichia coli cell.
 3. A composition according to claim 1, whereinthe plasmid is an enterotoxigenic E. coli plasmid in which the ST geneis inactivated or deleted.
 4. A composition according to claim 1,wherein the plasmid contains a deletion of all or part of the ST gene.5. A composition according to claim 1, wherein the bacterial cell isobtainable by a method comprising deletion of all or part of the ST genewith a suicide vector.
 6. A composition according to claim 1, whereinthe cell does not express heat labile toxin (LT).
 7. A compositionaccording to claim 1, wherein the cell does not express EAST1.
 8. Acomposition according to claim 1, wherein the cell does not express anantibiotic resistance gene.
 9. A composition according to claim 6,wherein the cell is obtainable by a method comprising site-directeddeletion or inactivation of the LT gene.
 10. A composition according toclaim 1, wherein the plasmid contains an element which enhances itsstability.
 11. A composition according to claim 10, wherein said elementis a toxin-antitoxin element or a recombinase recognition element.
 12. Acomposition according to claim 10, wherein the stability element isparDE or crs.
 13. A composition according to claim 1, wherein the cellis an Escherichia coli cell deposited with the European Collection ofCell Cultures (ECACC) under accession number 01090303, number 01090305,number 01090306, number 02082966, number 02082967 or number 02082968, ora descendent of a said cell.
 14. A composition according to claim 1,wherein the cell is attenuated by deletion or inactivation of at leastone aro gene and at least one omp gene.
 15. A composition according toclaim 1, wherein the cell is attenuated by deletion or inactivation ofat least one aro gene and the htrA gene.
 16. A composition according toclaim 1, wherein the cell is attenuated by deletion or inactivation ofeach of aroC, ompF and ompC.
 17. A composition according to claim 1,wherein the cell expresses a heterologous antigen.
 18. A compositionaccording to claim 17, wherein the heterologous antigen is an E. coliantigen.
 19. A composition according to claim 18, wherein theheterologous antigen is an E. coli colonization factor antigen (CFA).20. A composition according to claim 18, wherein the heterologousantigen is a non-toxic component or form of LT.
 21. A compositionaccording to claim 20, wherein the non-toxic component of LT is the Bsubunit.
 22. A composition according to claim 1, which further comprisesa cell which expresses colonization factor antigen CFA/II.
 23. Acomposition according to claim 1, which comprises: (i) said cell whichexpresses CFA/I; (ii) said cell which expresses CS5 and CS6; (iii) acell which expresses CS4 and CS6; (iv) a cell which expresses CS2 andCS3; and (v) a cell which expresses CS1 and CS3.
 24. A composition asclaimed in claim 23 wherein: (i) the cell which expresses CFA/I is thatdeposited with ECACC under accession number 01090303 or 02082967, or adescendent thereof; (ii) the cell which expresses CS5 and CS6 is thatdeposited with ECACC under accession number 01090305 or 02082968, or adescendent thereof; (iii) the cell which expresses CS4 and CS6 is thatdeposited with ECACC under accession number 01090306 or 02082966, or adescendent thereof; (iv) the cell which expresses CS2 and CS3 is thatdeposited with ECACC under accession number 01090304 or 02082964, or adescendent thereof; and (v) the cell which expresses CS1 and CS3 is thatdeposited with ECACC under accession number 01090302 or 02082965, or adescendent thereof.
 25. A method of immunization, comprisingadministering to a mammal the composition according to claim 1 [which:(a) induces an immune response to colonization factor CFA/I, andcomprises administering to a mammal a bacterial cell which expressescolonization factor antigen CFA/I from a native plasmid but does notexpress heat stable toxin (ST) or (b) induces an immune response tocolonization factor CS5 and/or colonization factor CS6, and comprisesadministering to a mammal a bacterial cell which expresses colonizationfactor antigen CS5 from a native plasmid and/or expresses colonizationfactor antigen CS6 from a native plasmid but does not express heatstable toxin (ST); wherein the cell is attenuated by deletion orinactivation of one or more gene(s) selected from the group consistingof aroA, aroC, aroD, aroE, pur, htrA, ompC, ompF, ompR, cya, crp, phoP,surA, rfaY, dksA, hupA, sipC and tipB].
 26. A method according to claim25, wherein said bacterial cell is an Escherichia coli cell.
 27. Amethod according to claim 25, wherein the plasmid is an enterotoxigenicE. coli plasmid in which the ST gene is inactivated or deleted.
 28. Amethod according to claim 25, wherein the plasmid contains a deletion ofall or part of the ST gene.
 29. A method according to claim 25, whereinthe bacterial cell is obtainable by a method comprising deletion of allor part of the ST gene with a suicide vector.
 30. A method according toclaim 25, wherein the cell does not express heat labile toxin (LT). 31.A method according to claim 25, wherein the cell does not express EAST1.32. A method according to claim 25, wherein the cell does not express anantibiotic resistance gene.
 33. A method according to claim 25, whereinthe cell is obtainable by a method comprising site-directed deletion orinactivation of the LT gene.
 34. A method according to claim 25, whereinthe plasmid contains an element which enhances its stability.
 35. Amethod according to claim 34, wherein said element is a toxin-antitoxinelement or a recombinase recognition element.
 36. A method according toclaim 34, wherein the stability element is parDE or crs.
 37. A methodaccording to claim 25, wherein the cell is an Escherichia coli celldeposited with the European Collection of Cell Cultures (ECACC) underaccession number 01090303, number 01090305, number 01090306, number02082966, number 02082967 or number 02082968; or a descendent of a saidcell.
 38. A method according to claim 25, wherein the cell is attenuatedby deletion or inactivation of at least one aro gene and at least oneomp gene.
 39. A method according to claim 25, wherein the cell isattenuated by deletion or inactivation of at least one aro gene and thehtrA gene.
 40. A method according to claim 25, wherein the cell isattenuated by deletion or inactivation of each of aroC, ompF and ompC.41. A method according to claim 25, wherein the cell expresses aheterologous antigen.
 42. A method according to claim 41, wherein theheterologous antigen is an E. coli antigen.
 43. A method according toclaim 42, wherein the heterologous antigen is an E. coli colonizationfactor antigen (CFA).
 44. A method according to claim 42, wherein theheterologous antigen is a non-toxic component or form of LT.
 45. Amethod according to claim 44, wherein the non-toxic component of LT isthe B subunit.
 46. A method according to claim 25, which furthercomprises administering a cell which expresses colonization factorantigen CFA/II.
 47. A method according to claim 25, which comprisesadministering: (i) said cell which expresses CFA/I; (ii) said cell whichexpresses CS5 and CS6; (iii) a cell which expresses CS4 and CS6; (iv) acell which expresses CS2 and CS3; and (v) a cell which expresses CS1 andCS3.
 48. A method as claimed in claim 47 wherein: (i) the cell whichexpresses CFA/I is that deposited with ECACC under accession number01090303 or 02082967, or a descendent thereof; (ii) the cell whichexpresses CS5 and CS6 is that deposited with ECACC under accessionnumber 01090305 or 02082968, or a descendent thereof; (iii) the cellwhich expresses CS4 and CS6 is that deposited with ECACC under accessionnumber 01090306 or 02082966, or a descendent thereof; (iv) the cellwhich expresses CS2 and CS3 is that deposited with ECACC under accessionnumber 01090304 or 02082964, or a descendent thereof; and (v) the cellwhich expresses CS1 and CS3 is that deposited with ECACC under accessionnumber 01090302 or 02082965, or a descendent thereof.